Method for extracting quantitative information relating to interactions between cellular components

ABSTRACT

A method is described to assay for protein interactions in living cells, e.g. by the introduction of two heterologous conjugates into the cell. The method uses the measurement of cellular distribution of a detectable component (e.g. a GFP-labelled fluorescent probe) to indicate the presence or absence of an interaction between that component and a second component of interest. The method uses the knowledge that certain components can be stimulated to redistribute within the cell to defined locations. Inducible redistribution systems make it possible to determine if specific interactions occur between components. Inducible systems are described where it is demonstrated that the redistribution stimuli are essentially “null”, in that they affect no other system in the cell during the assay period, other than the component whose redistribution can be induced. Also described is an extraction buffer which is useful in high throughput screening for drugs which affect the intracellular distribution of intracellular components. The extraction buffer comprises a cellular fixation agent and cellular permeabilisation agent. Optimising the composition of the extraction buffer and its application to various cell types is described.

FIELD

[0001] The present invention relates to measurement of interactionsbetween two components wherein the two components are present in a cell,and where both components are most usually wholly or mainlyproteinaceous in composition (i.e. the interaction is a protein-proteininteraction, or protein-protein binding event). This is presentlyreferred to as “GFP assisted Readout of Interacting Proteins (GRIP)”

[0002] The present invention also relates to an extraction buffer usedin high throughput screening for drugs that affect the intracellulardistribution of intracellular components. The extraction buffercomprises a cellular fixation agent and a cellular permeabilizationagent.

BACKGROUND

[0003] The interaction of proteins with each other and with othercellular components is an intrinsic part of nearly every cellularprocess, and this is especially true of intracellular signallingsystems. Information is passed through and between signalling systems bya series of such interactions. In order to study the function of aprotein, a practical strategy is first to identify the components thatit interacts with. Most of these will be other proteins—sometimes of thesame species, but most often a very different type and with verydifferent functional characteristics.

[0004] The identification of novel interactions is a very rapidlygrowing area of research in cell biology and signal transduction. Anotable feature of recent discoveries in this area is the specificitywith which partners interact, equalling or exceeding the degree ofspecificity commonly seen in ligand-receptor interactions seen at thecell surface. Identification of interacting species brings with it theopportunity to identify novel signalling interactions that may assistgreatly in the functional characterisabon of proteins involved incellular signalling. In addition the method will be applicable to thedevelopment of new pharmaceutical agents capable of disrupting orengaging partners in an interaction.

[0005] Compounds with this mechanism of action will be able to modulatethe flow of information through signalling pathways, and in so doingfind application in very many areas of human and animal health care.Since such compounds will be inherently very selective and have theiraction without the need for gross inhibition of catalytic activity, itcan be expected that therapies based on their use will not carry withthem the problems of poor specificity and damaging side effects commonlyassociated with more traditional active-site inhibitors.

[0006] Existing methods for identification of interacting species can bedivided into two groups: First are those methods that can only work withmore or less purified components brought together in vitro, such assurface plasmon resonance (evanescent wave methods), protein massspectroscopy, fluorescence correlation spectroscopy and anisotropymeasurements all with the common feature that the components of interestare isolated from the cellular context. The second group includes allmethods designed to work within living cells. Of these, many have beendeveloped to work in yeast cells (yeast two hybrid, reverse yeast twohybrid and variations thereof) but some have been adapted for use inmammalian cell systems. Cellular methods for detection of proteininteractions have been well reviewed by Mendelsohn, A. R., Brent, R.(1999) (Science 284(5422):1948). Many of these methods are descendantsof the conventional two-hybrid methods, where transcriptional activityis initiated by the bringing together of bi-partite transcriptionfactors through the interaction of attached “bait” and “prey”components, while other methods rely on reconstitution of a biochemicalfunction in vivo. Rossi et al. (2000) (Trends in Cell Biology10:119-122) have thus developed a mammalian cell-based protein-proteininteraction assay where the read-out is not transcriptional butreconstitution of a mutated beta-galactosidase enzyme. Uponreconstitution of the tetrameric enzyme, enzymatic activity can bemonitored. In addition methods for monitoring protein-proteininteractions that are based on an optical read-out i.e. fluorescenceresonance transfer (FRET), or coincidence analysis (a variant offluorescence correlation spectroscopy), or fluorescence lifetimechanges. The last three categories are more normally applied undersimplified in vitro conditions, but attempts are being made to move theminto the more complex environment of the living cell.

[0007] Recently Tobias Meyer reported (WO00/17221) a method wherein twoheterologous conjugates are introduced into a cell. The firstheterologous conjugate comprises the first protein of interestconjugated to a detectable group (e.g. GFP). The second heterologousconjugate comprises a second protein of interest conjugated to a proteinthat specifically binds to an internal structure within the cell uponstimulation with phorbol ester. When the second protein is bound to aninternal structure within the cell, with a known distribution, bindingbetween the two proteins of interest can be visualised as the detectablegroup will be located bound to internal structure within the cell.

[0008] Proteins(-GFP) that are not “anchored” in an intracellularorganelle or compartment, but are more or less mobile in the cytosol,will diffuse into the surrounding medium upon cell permeabilization, ata rate largely governed by the degree of permeabilisation imposed. It isdifficult to control the release of cellular contents bypermeabilization since detergents have the tendency to not only disruptthe membrane but also over time disrupt intracellular components. Besidethis, damaging the membrane will start some uncontrolled proteaseactivity with the same unintentional result. This phenomenon is alsoseen when using non detergent permeabilization (eg. Digitonin). Fixativeagents are commonly used to preserve structural integrity in cellsduring the processes necessary to prepare biological material formicroscopy. Fixatives aimed at preserving or stabilising proteinstructures within cells can be divided into two groups; those thatcoagulate proteins, such as organic acids or alcohols (e.g. acetic acid,ethanol), and those that cross-link proteins together into an insolublenetwork, such as the aldehydes (e.g. formaldehyde or glutaraldehyde).The rate of fixation by such agents is governed by their rate ofpenetrance into cells together with the rate of chemical cross-linkingor coagulation that they can achieve. The processes and methods ofcellular fixation have been thoroughly studied and described in thescientific literature (see, for example, Fixation for ElectronMicroscopy by M. A. Hayat, 1981, Academic Press, New York).

[0009] Translocation usually involves changes in the effective mobilityof at least one protein within the cell, either through changes ininteraction between that component and an anchored (or effectivelynon-mobile) component or through a change in location orcompartmentation of the component, for example a transfer from theextra-nuclear cytoplasm into the nucleus itself. Changes in interactionor compartmentation constitute translocation. Redistribution™ is the artof making translocation a measurable event (WO98/45704). Translocationmay involve change in mobility or compartmentation of a component towhich the component of interest is attached, for example a motor proteinby which the component of interest is carried. Some translocationsinvolve sub-microscopic distances, for instance the interaction betweena soluble signaling protein in the cytoplasm and an adjacent actinfilament or intracellular membrane. The mobility of the previouslysoluble component is greatly changed in such a translocation, but anychange in its position in space may be unresolvable by microscopicmeans. In this case, it would be desirable to separate mobile fromimmobile forms of the protein of interest in order to reveal theredistribution signal.

[0010] Usually redistribution™ is measured by “imaging”, which is verytime consuming and inconvenient for drug screening in HT format.Redistribution™ assays often comprise cell lines stably expressing aparticular protein of interest, (most often that protein being anengineered protein fusion between the protein of interest and aluminescent protein such as GFP). In such cells, it may be the case thatthe component of interest is overexpressed to some degree relative tothe other components in the cell with which it should interact. This canlead to a masking of the translocation event by excess amounts of theprotein of interest that cannot physically interact with the limitednumber of partner components available in the cell. In such cases,removal of the excess component of interest may be sufficient to unmaskthe translocation that has occurred.

[0011] Fixation of the cells before permeabilization is never successfulin such cases, since all proteins become effectively immobilized toapproximately the same degree under the influence of the fixative, andhence cannot be differentially removed or washed away from the cell.

SUMMARY OF THE INVENTION

[0012] The interaction between Sos and Grb2 can be measured in variousways. As shown in the Example 7, cells co-transfected with a PDE4A4-SosAconjugate and with a Grb2-EGFP conjugate illustrate the binding betweenSosA and Grb2. In Example 8 the conjugates are PKAcat-hGrb2 andEGFP-Sos-Cterm. The advantage of the PKAcat conjugates is that nospecial treatment is necessary in order to visualise interaction, aslong as levels of cAMP within cells remain sufficiently low so that PKAis predominately attached to it regulatory subunits. In Example 9 theconjugates are Cys1-Grb2 and EGFP-Sos-Cterm. The possible advantage ofthe Cys1 conjugates are that they will take one component of a pair tothe plasma membrane when treated with PMA or some other activationstimulus. This special location may be necessary in order to activateinteraction of the components being studied.

[0013] Example 3 describes a generic way to produce cell lines byco-transfection of two plasmids, each expression a heterologousconjugate. As illustrated in this example, by using PDE4A4 based anchorprobes as anchor-proteins an average of two dense aggregates is formedby treatment with rolipram. These aggregates can by detected directlywith an antibody directed against the unique C-terminal peptide sequenceof PDE4A. The dense aggregates (the spots) will disappear when eitherrolipram is removed or competed against with one of a particular classof PDE4 inhibitors (e.g. RP73401). It is important to note that thetreatment with rolipram does not affect the levels of cAMP in the cells(Example 14).

[0014] Example 4 describes that using PKAcata based anchor probes asanchor-proteins aggregates are formed in the cytoplasm when theconcentration of cytoplasmic cAMP is low. These aggregates can bydetected directly with an antibody directed against PKAc or against thePKA regulatory subunit. These aggregates disperse into the cytoplasmwhen cAMP concentrations are elevated.

[0015] Example 5 describes that using PDE4A1 based anchor probes asanchor-proteins, small perinuclear spots are formed in the cytoplasm inotherwise untreated cells. PDE4A1 spots can by detected directly with anantibody directed against the unique C-terminal peptde sequence ofPDE4A. These spots disperse into the cytoplasm when rolipram is added tothe cells.

[0016] Example 6 describes that using Cys1 domain based anchor probes asanchor-proteins redistributes to the plasma membrane when activated e.g.by treatment with PMA. The localisation of the Cys1 domain can beconfirmed with antibodies directed against the myc or flag antigens(when myc or flag sequences are included in the genetic construct usedto transfect the cells).

[0017] Application of fixative and permeabilisation simultaneously, asdescribed in this document, aims to preserve local concentrations of theanchored, diffusionally restricted or largely immobile form of theprotein of interest while allowing freely mobile or unanchored forms tobe released from the cell. It is expected that the process of cellpermeabilisation, especially by detergents, will in time remove evenrelatively immobile, anchored or compartmentalised proteins from thecell, and hence a balance must be found between the rate ofpermeabilisation and solubilisation caused by the detergent agent, andthe rate at which cross-linking or fixation takes place. This balancemay be achieved by careful selection of the fixative andpremeabilisation agents, controlling the relative concentations of theseagents, also by controlling the physical and chemical conditions underwhich the agents work (pH, osmolarity or ionic strength, temperature).

[0018] In order to control the permeabilization to a level that allows“floating” proteins to leave the cells without disrupting intracellularorganelles and compartments, cells are slightly chemically fixed duringthe permeabilization process. The purpose is to just let the “non bound”proteins leave the cells and then have the rest chemically fixed. Thepresent application discloses how complex intracellular movements withinthe cell can be measured easily. In Example 10, the change in mobilityof BAD caused by the binding between the 14-3-3 protein and BAD ismeasured using a PDE4A4-14-3-3 and an EGFP-BAD fusion. The PDE4A4-14-3-3protein is stuck in spots within the cell due to previous treatment withrolipram. By extracting the soluble (or mobile) BAD, fused to GFP, witha buffer comprising a combination of a cellular permeabilization agentand a cellular fixation agent, only the immobile GFP is left in thecell, and the change in mobility of BAD caused by binding to the 14-3-3protein, can be read as a change in fluorescence intensity. Example 11shows the same principle applied to the Redistribution™ of NFkB(p65)from cytosol to nucleus upon stimulation of NFkB. The optimization ofthe extraction buffer, to identify the optimal ratio between thecellular fixation agent and the cellular permeabilisation agent isillustrated in Example 12.

DETAILED DISCLOSURE

[0019] The redistribution-trap method, subject of this application, isdifferent from any of the above-mentioned methods in that it utilisespositional information from PDE4 and in one aspect distribution of aGFP-labelled fluorescent probe in a cell, to indicate the presence orabsence of an interaction between specific components. That it does soin the complex environment of the cell, which allows for the influenceof factors which may modulate an interaction in the same way as wouldhappen in the native system, adds important physiological relevance tothe method. Since it is based on non-destructive fluorescence imaging,meaning that the cells can be live and active whilst being monitored,and since it is based on non-disturbing treatment with e.g. rolipram,the redistribution-trap method also allows transient or conditionalinteractions to be monitored. Transient or conditional interactions mayoccur when components are phosphorylated or otherwise modified duringtheir cycle of operation (e.g. transmission of a signal), and suchmodifications are common amongst components of intracellular signallingpathways. As the method does not rely on covalent interactions nor thatthe components need have a specific orientation upon interaction, themethod is very sensitive and allow for measurement of even low affinityinteractions.

[0020] One aspect thus relates to a method for detecting if a compoundmodulates an intracellular protein interaction comprising the steps of:

[0021] (a) providing a cell that contains two heterologous conjugates,the first heterologous conjugate comprising a first protein of interestconjugated to a detectable group, the second heterologous conjugatecomprising a second protein of interest conjugated to an anchor proteinthat can specifically bind to an internal structure within the cell;

[0022] (b) detecting the intracellular distribution of the detectablegroup an intracellular distribution of said detectable group mimickingthe intracellular distribution of the anchor-protein being indicative ofbinding between the two proteins of interest;

[0023] (c) repeating step (b) with and without the compound; a change inintracellular distribution of the detectable group with and without thecompound being indicative of the compound modulating said proteininteraction.

[0024] The redistribution-trap method according to the present inventionmakes use of the fact that many signalling components redistributewithin the cell to specific locations upon specific stimuli ortreatments. If those components can be labelled in some way to make themvisible in the cell, their location can be monitored and measured by anumber of image-based techniques. Since imaging techniques arenon-destructive, they allow measurements to be made on living cells,hence active processes can be followed over time if that is required—asmay be the case when transient events need to be monitored. Thisapplication details how the knowledge that a particular component willredistribute upon receiving a certain stimulus (the “anchor” stimulus)can be harnessed, to create a system to explore interactions betweenintracellular components. If a component is known to distribute to aknown cellular location upon a certain stimulus, another component (the“bait”) may be covalently attached to the first (the “anchor”) and,given the appropriate anchor stimulus, will be expected to assume thesame distribution in the cell as the anchor component to which it isattached. A further component (the “prey”), which is expected tointeract with the bait component, is introduced into the same cell. Theprey component is labelled in some way to make it visible in the cell.If an interaction occurs between bait and prey (perhaps requiring afurther interaction “stimulus”), then the prey component also takes upthe same distribution within the cell as the anchor-bait component, butonly if the appropriate anchor stimulus has also been applied. Usingthis system it is therefore possible to distinguish between specificbait-prey interactions and any other condition affecting thedistribution of the detectable prey component. The redistributon-trapmethod is therefore able to impose a gross redistribution uponinteracting components within the cell, even if the components inisolation would not normally display an appreciable redistribution aspart of their functional cycle. Anchor systems can be designed toachieve redistribution to compartments or locations within cells wherethe interacting components may experience the influences that wouldnormally be required to modulate the interaction between thosecomponents. As an example, some components may normally require to bephosphorylated or dephosphorylated by enzymes sequestered in the planeof the plasma membrane—for such components it would be appropriate foran anchor system to be chosen such that the anchor stimulusredistributed the anchor probe to the plasma membrane, to allow theinteracting components to be appropriately modified. An example of suchan anchor system would be one based on the Cys1 domain of PKCY. Themethod also allows for targeting interactions to different locationswithin the cell with the purpose of studying whether location specificconditions are necessary for the interaction to occur. In one embodimentone or both interacting components are targeted to the nuclearcompartment. In another embodiment, one or both interacting componentsare targeted to mitochondria outer or inner membranes. In anotherembodiment, one or both interacting components are targeted to differentregions of Golgi bodies. In another embodiment, one or both interactingcomponents are targeted to focal adhesion complexes. In anotherembodiment, one or both interacting components are targeted tocytoskeletal structures such as F-actin strands or microtubular bundles.In another preferred embodiment, one or both interacting components aretargeted to the plasma membrane. In another preferred embodiment, one orboth interacting components are targeted to cytoplasmic granules oraggregates such as those formed by PDE4A4 in the presence of rolipram.

[0025] Thus, a specific embodiment of the present invention relates to amethod wherein the specific binding to the internal structure within thecell is induced by an anchor stimulus that by itself has littlelikelihood of stimulating or inhibiting signalling activity within thecell of interest.

[0026] Due to the strong anchoring response of PDE4A4 to rolipram andother specific PDE4 inhibitors such as RS25344, PDE4A4 is most preferredas the anchoring species. Furthermore, it seems that the attachment ofPDE4A4 to it's anchor site is not affected by he presence of additionalparts attached to the C-terminal of the PDE4A4 molecule, here theseattachments can be of very variable size (from less than 10 kDa to atleast 150 kDa). Thus, a specific embodiment of the present inventionrelates to a method as described above wherein the specific binding isinduced by addition of the anchor stimulus rolipram and wherein theanchor protein is PDE4A4.

[0027] The particular utility of stimulus-induced distributions, such asthose that are based on PDE4A4 anchors or PKCαCys1 domains, is that inone and the same cell it is possible to switch on a distinctivedistribution where previously there was none. This not only guarantees,in advance, that the distinctive distribution is purely a result ofspecific interaction between anchored and detectable components (theanchor component responding to the stimulus is “invisible” unlessdecorated by the detectable component), but also guarantees that thisinteraction will give a signal that is measurable by the assay equipmentconfigured to detect the specific and expected distribution of theanchor component. In effect this latter point means that many differentinteractions can be measured and assayed without the need to reconfigurethe measuring equipment or the assay method. Also, the inducing stimulusprovides a reference compound in screening assays by which the maximumand minimum expected signals for an assay can be determined.

[0028] Many signalling components have a specific location within thecell and redistribute upon specific stimuli or treatments. If thosecomponents can be labelled in some way to make them visible in the cell,their location can be monitored and measured by a number of image-basedtechniques. Since imaging techniques are non-destructive, they allowmeasurements to be made on living cells, hence active processes can befollowed over time if that is required—as may be the case when transientevents need to be monitored. This application details how the knowledgethat a particular component will redistribute upon receiving a certainstimulus (the “anchor” stimulus) can be harnessed, to create a system toexplore interactions between intracellular components. If a component isknown to have an inherently distinctive cellular distribution , anothercomponent (the “bait”) may be covalently attached to the first (the“anchor”) and, without any anchor stimulus, will be expected to assumethe same distribution in the cell as the anchor component to which it isattached. A further component (the “prey”), which is expected tointeract with the bait component, is introduced into the same cell. Theprey component is labelled in some way to make it visible in the cell.If an interaction occurs between bait and prey (perhaps requiring afurther “interaction stimulus”), then the prey component also takes upthe same distribution within the cell as the anchor-bait component, evenwithout application of an anchor stimulus due to the specific locationin the first place. Further, if the inherent distinctive distribution ofthe anchor component will be dissolved by application of an anchorstimulus, then such a system is useful to distinguish between specificbait-prey interactions and any other condition affecting thedistribution of the detectable prey component. The redistribution-trapmethod is therefore able to impose a gross redistribution uponinteracting components within the cell, even if the components inisolation would not normally display an appreciable redistribution aspart of their functional cycle. Anchor systems can be designed toachieve redistribution to compartments or locations within cells wherethe interacting components may experience the influences that wouldnormally be required to modulate the interaction between thosecomponents. As an example, the spot like cytoplasmic distribution of PKAcan be dissolved by addition of forskolin; the spot like cytoplasmicdistribution of PDE4A1 can be dissolved by addition of rolipram; thespot like nuclear distribution of histonedeacetylase 5 (HDAC5) isdissolved by Trichostatin A (TSA); or the specific binding of the anchorprotein is PLC-delta to the cell membrane is dissolved by addition ofATP and distributed within the cytoplasm.

[0029] For these systems where a distinctive distribution is dispersedor dissolved by a specific stimulus (the anchor stimulus), such as thosebased on PDE4A1, PKAcat, PLCdelta or HDAC5, the stimulus provides ameans to check whether that distinct distribution is the result ofinteraction between anchored and detectable components, or results fromsome inherent tendency of the detectable component to assume thatdistinct distribution within the cell. The check for this can be made inone and the same cell that the assay for interaction is measured. Aswith stmulus-induced distributions, systems with an initial distinctdistribution of the anchor component share the advantages of being ableto assay many different interactions with one configuration of equipmentand assay protocol. The anchor stimulus in each case again provides areference compound in screening assays by which the maximum and minimumexpected signals for an assay can be determined. The particularadditional advantage of those systems where the anchor stimulusdisperses a distribution is that no pretreatment or co-treatment ofcells with distribution stimuli is necessary during the assay procedure,precluding any possibility that the anchor stimulus may interfere,directly or indirectly, with the interaction that is being tested.

[0030] The term “compound” is intended to indicate any sample, which hasa biological function or exerts a biological effect in a cellularsystem. The sample may be a sample of a biological material such as asample of a body fluid including blood, plasma, saliva, milk, urine, ora microbial or plant extract, an environmental sample containingpollutants including heavy metals or toxins, or it may be a samplecontaining a compound or mixture of compounds prepared by organicsynthesis or genetic techniques. The compound may be small organiccompounds or biopolymers, including proteins and peptides.

[0031] The compound to be tested can be regarded as a specialinteraction stimulus.

[0032] Numerous cell systems for transfection exist. A few examples areXenopus oocytes or insect cells, such as the sf9 cell line, or mammaliancells isolated directly from tissues or organs taken from healthy ordiseased animals (primary cells), or transformed mammalian cells capableof indefinite replication under cell culture conditions (cell lines).However, it is preferred that the cells used are mammalian cells. Thisis due to the complex biochemical interactions specific for each celltype. The term “mammalian cell” is intended to indicate any living cellof mammalian origin. The cell may be an established cell line, many ofwhich are available from The American Type Culture Collection (ATCC,Virginia, U.S.A.) or similar Cell Culture Collections. The cell may be aprimary cell with a limited life span derived from a mammalian tissue,including tissues derived from a transgenic animal, or a newlyestablished immortal cell line derived from a mammalian tissue includingtransgenic tissues, or a hybrid cell or cell line derived by fusingdifferent cell types of mammalian origin e.g. hybridoma cell lines. Thecells may optionally express one or more non-native gene products, e.g.receptors, enzymes, enzyme substrates, prior to or in addition to thefluorescent probe. Preferred cell lines include but are not limited tothose of fibroblast origin, e.g. BHK, CHO, BALB, NIH-3T3 or ofendothelial origin, e.g. HUVEC, BAE (bovine artery endothelial), CPAE(cow pulmonary artery endothelial), HLMVEC (human lung microvascularendothelial cells), or of airway epithelial origin, e.g. BEAS-2B, or ofpancreatic origin, e.g. RIN, INS-1, MIN6, bTC3, aTC6, bTC6, HIT, or ofhematopoietic origin, e.g. primary isolated human monocytes,macrophages, neutrophils, basophils, eosinophils and lymphocytepopulations, AML-14, AML-193, HL-60, RBL-1, U937, RAW, JAWS, or ofadipocyte origin, e.g. 3T3-L1, human pre-adipocytes, or ofneuroendocrine origin, e.g. AtT20, PC12, GH3, muscle origin, e.g. SKMC,A10, C2C12, renal origin, e.g. HEK 293, LLC-PK1, or of neuronal origin,e.g. SK-N-DZ, SK-N-BE(2), HCN-1A, NT2/D1.

[0033] The examples of the present invention are based on CHO cells.Therefore fibroblast derived cell lines such as BALB, NIH-3T3 and BHKcells are preferred.

[0034] It is preferred that the two heterologous conjugates areintroduced into the cell as plasmids, e.g. two individual plasmids mixedupon application to cells with a suitable transfection agent such asFugene (see Example 3, Example 4, Example 5 and Example 6) so thattransfected cells express and integrate both the first and the secondheterologous conjugates simultaneously. Many other means forintroduction of one or both of the conjugates are evenly feasible e.g.electroporation, calcium phosphate precipitate, microinjection,adenovirus and retroviral methods, bicistronic plasmids encoding bothconjugates etc.

[0035] Throughout the present invention, the term “protein” should havethe general meaning. That includes not only the translated product, butalso chemically synthesised proteins. For proteins translated within thecell, the naturally, or induced, post-translational modifications suchas glycosylation and lipidabon are expected to occur and those productsare still considered proteins. The term intracellular proteininteraction has the general meaning of an interaction between twoproteins, as described above, within the same cell. The interaction isdue to non-covalent forces between the protein components, most usuallybetween one or more regions or domains on each protein whosephysico-chemical properties allow for a more or less specificrecognition and subsequent interaction between the two protein,components involved. In a preferred embodiment, the intracellularinteraction is a protein-protein binding.

[0036] The luminophore allows the spatial distribution of the componentto be visualised and/or recorded by emitting light. In a preferredaspect of the invention, the luminophore is capable of beingredistributed in substantially the same manner as the component. In yetanother embodiment of the invention, the luminophore is capable of beingquenched upon spatial association with a component which isredistributed by modulation of the pathway, the quenching being measuredas a change in the intensity or lifetime of the luminescence. In apreferred aspect, the luminophore is a fluorophore. In a preferredembodiment of the invention, the luminophore is a polypeptide encoded byand expressed from a nucleotide sequence harboured in the cell or cells.E.g. the luminophore is a part of a hybrid polypepbde comprising afusion of at least a portion of each of two polypeptides one of whichcomprises a luminescent polypeptide and the other one of which comprisesthe component. As the examples are carried out with GFP, GFP isespecially preferred.

[0037] In the present context, the term “green fluorescent protein”(GFP) is intended to indicate a protein which, when expressed by a cell,emits fluorescence upon exposure to light of the correct excitationwavelength (e.g. as described by Chalfie, M. et al. (1994) Science 263,802-805). Such a fluorescent protein in which one or more amino acidshave been substituted, inserted or deleted is also termed “GFP”. “GFP”as used herein includes wild-type GFP derived from the jelly fishAequorea victoria, or from other members of the Coelenterata, such asthe red fluorescent protein from Discosoma sp. (Matz, M. V. et al. 1999,Nature Biotechnology 17: 969-973) or fluorescent proteins from otheranimals, fungi or plants, and modifications of GFP, such as the bluefluorescent variant of GFP disclosed by Heim et al. (Heim, R. et al.,1994, Proc.Natl.Acad.Sci. 91:26, pp 12501-12504), and othermodifications that change the spectral properties of the GFPfluorescence, or modifications that exhibit increased fluorescence whenexpressed in cells at a temperature above about 30° C. described inPCT/DK96/00051, published as WO 97/11094 on Mar. 27, 1997 and herebyincorporated by reference, and which comprises a fluorescent proteinderived from Aequorea Green Fluorescent Protein or any functionalanalogue thereof, wherein the amino acid in position 1 upstream from thechromophore has been mutated to provide an increase of fluorescenceintensity when the fluorescent protein of the invention is expressed incells. Preferred GFP variants are F64L-GFP, F64L-Y66H-GFP F64L-S65T-GFP,F64L-E222G-GFP. One especially preferred variant of GFP for use in allthe aspects of this invention is EGFP (DNA encoding EGFP which is aF64L-S65T variant with codons optimized for expression in mammaliancells is available from Clontech, Palo Alto, plasmids containing theEGFP DNA sequence, cf. GenBank Acc. Nos. U55762, U55763). Anotherespecially preferred variant of GFP is F64L-E222G-GFP. As used in theexamples, the detectable group preferably is a green fluorescent protein(GFP). The GFP is preferably selected from the group consisting of GFPshaving the F64L mutation as defined herein such as F64L-GFP,F64L-Y66H-GFP, F64L-S65T-GFP, EGFP, and F64L-E222G-GFP. The GFP is N- orC-terminally tagged, optionally via a peptide linker, to thebiologically active polypeptide or a part or a subunit thereof.

[0038] In an alternative embodiment the detectable groups is labelledwith chemical fluorophores either in situ or by microinjection orotherwise introduced into cells. In yet another embodiment thedetectable group comprises an epitope for antibodies, which arethemselves detectable by other methods, either because they are taggedwith a fluorophore, or may be detected by a biotin-streptavidinlabelling method, or by secondary antibodies labelled with fluorophoresetc. Examples of such epitopes detectable tag such as the myc or flagantigens, which may then be detected with antibodies, which arethemselves detectable by other methods, either because they are taggedwith a fluorophore, or may be detected by a biotin-streptavidinlabelling method, or by secondary antibodies labelled with fluorophoresetc.

[0039] Internal structure as used herein refers to a separate, discreet,identifiable component contained within a cell. Such internal structuresare, in general, anatomical structures of the cell in which they arecontained. Examples of internal structures include both structureslocated in the cytosol or cytoplasm outside of the nucleus (also calledcytoplasmic structures) and structures located within the nucleus(nuclear structures). The nucleus itself including the nuclear membraneis an internal structure.

[0040] The recording of the detectable group will vary with thedetectable group chosen. For example, when GFP is used as a detectablegroup the emitted light can be measured with various apparatus known tothe person skilled in the art. Typically such apparatus comprises thefollowing components: (a) a light source, (b) a method for selecting thewavelength(s) of light from the source which will excite theluminescence of the luminophore, (c) a device which can rapidly block orpass the excitation light into the rest of the system, (d) a series ofoptical elements for conveying the excitation light to the specimen,collecting the emitted fluorescence in a spatially resolved fashion, andforming an image from this fluorescence emission (or another type ofintensity map relevant to the method of detection and measurement), (e)a bench or stand which holds the container of the cells being measuredin a predetermined geometry with respect to the series of opticalelements, (f) a detector to record the light intensity, preferably inthe form of an image, (g) a computer or electronic system and associatedsoftware to acquire and store the recorded information and/or images,and to compute the degree of redistribution from the recorded images.

[0041] In a preferred embodiment of the invention the apparatus systemis automated. In one embodiment the components in d and e mentionedabove comprise a fluorescence microscope. In one embodiment thecomponent in f mentioned above is a CCD camera. In one embodiment thecomponent in f mentioned above is an array of photomultipliertubes/devices.

[0042] In one embodiment of the invention the actual fluorescencemeasurements are made in a standard type of fluorometer for plates ofmicrotiter type (fluorescence plate reader).

[0043] In one embodiment the optical scanning system is used toilluminate the bottom of a plate of microtiter type so that atime-resolved recording of changes in luminescence or fluorescence canbe made from all spatial limitations simultaneously.

[0044] In one embodiment the image is formed and recorded by an opticalscanning system. In a preferred embodiment the actual luminescence orfluorescence measurements are made in a FLIPR™ instrument, commerciallyavailable from Molecular Devices, Inc.

[0045] The quantitative information which is indicative of the degree ofthe cellular response to the influence or the result of the influence onthe intracellular pathway is extracted from the recording or recordingsaccording to a predetermined calibration based on responses or results,recorded in the same manner, to known degrees of a relevant specificinfluence. Hereby the degree of redistribution caused by an influence isexpressed as the dose of a relevant specific influence causing samedegree of cellular response. By testing a unknown influence, e.g. newchemical entities or chemicals without known effect on theredistribution of the cellular component a screening assay for drugswith effect on redistribution is achieved.

[0046] Based on these scientific and intellectual findings, the presentinvention can among other things be useful to:

[0047] Create cellular assays to monitor interactions between cellularcomponents at the intermolecular level. Create cellular assays tomonitor transient interactions between cellular components.

[0048] Create cellular assays to monitor conditional interactionsbetween cellular components.

[0049] Create cellular assays to monitor interactions between componentsthat have low affinity for one another.

[0050] Create cellular test systems in which the mobility of specificmolecular components, for example a species of signalling molecule, canconditionally be restricted (locked down) to achieve a functionalknockout of activity for that species.

[0051] Create cellular test systems in which the mobility of specificmolecular components, for example a species of signalling molecule, canconditionally be released (dispersed) to achieve a functional knock-inof activity for that species.

[0052] Create a cellular system where interaction events betweenspecific components are restricted to a specific location

[0053] Examples of uses are:

[0054] Cellular assays to find inhibitors of interactions

[0055] Cellular assays to find activators of interactions

[0056] Cellular assays to identify novel binding partners to anyspecific cellular component through screening of cDNA libraries.

[0057] Cellular assays to automatically screen active compounds fortheir ability to penetrate the cell membrane to provide an indication oflikely bioavailability.

[0058] Cellular assays to automatically screen compounds for stabilityin the cellular environment to provide an indication of compoundscellular metabolism and excretion over the period of the assay.

[0059] Cellular test systems to investigate the function of specificcellular components.

[0060] Cellular assays to identify the signalling pathways used byorphan receptors and/or orphan ligands.

[0061] Cellular assays in high throughput screening for interactionmodulators.

[0062] Some features of the present invention:

[0063] Mammalian cell assays provide physiologically relevant contextfor interactions, to allow for the influence of factors which maymodulate an interaction as they would in the native system, for instanceas in cells in the intact human system.

[0064] The redistribution-trap idea is compatible with a variety offluorescence imaging methods, using either live or chemically fixedcells.

[0065] The response of any of the assays based on theredistribution-trap idea can be monitored either continuously as asequential series of measurements over time, to generate a time coursefor a response, or by single end-point measurement. Time coursemeasurements require live cells throughout. End point measurements canbe made on either live or chemically fixed cells.

[0066] The method is amenable to low as well as high throughputmeasurements.

[0067] The method is useful in mammalian cells. Mammalian cells, unlikecells of plant or fungal origin such as yeast cells, have no cell wall.A cell wall can prevent certain compounds from entering the cell.

[0068] The measurement of protein interactions described above is idealfor identification/screening of compounds modulating such interactions.In order to carry out such assays, and other assays in High Throughput,one aspect of the present invention relates to a method for measuring achange in mobility of a cellular component caused by an influence, themethod comprising:

[0069] (a) contacting or incubating cells with and without theinfluence, the cells comprising a luminophore coupled to the cellularcomponent;

[0070] (b) adding extraction buffer to the cells of step (a), theextraction buffer comprising a cellular fixation agent and a cellularpermeabilisation agent; and

[0071] (c) measuring the light emitted from the luminophore from cellsof step (b); wherein a difference between light emitted from the cellswith and without the influence indicates a difference in the mobility ofthe cellular component caused by the influence. The principle is toremove luminophore-component couplings that has either moved or notmoved upon stimulation.

[0072] One major advantage of the present invention is that changes inmobility can be measured as a change in light intensity. As will beillustrated below, and as described in the examples, this techniqueallows Redistribution™ to be detected as a fluorescence intensitychange.

[0073] A variety of instruments exist to measure light intensity. In apreferred aspect of the present invention, wherein the luminophore isGFP, the instrument for measuring the light emitted from the luminophoreis a FLIPR (Molecular Devices). In an alternative embodiment, the lightemitted from the luminophore is measured on a plate reader. Thistechnique has improved Redistribution™ assays like the NFkB assay(Example 11) and TRAP assays like PDE4A4-trap (Example 10 and Example12) from a screen time of 4 hrs per plate (imaging) to app. 30 sec. perplate (that is 480 times faster).

[0074] Throughout this application the change in mobility ischaracterized in that the component is substantially immobile eitherprior to contact or incubation with the influence or after contact orincubation with the influence. Substantially immobile is defined as thecomponent will not move outside of the detection field during theextraction procedure. The detection field, when the cells are washedafter extraction, will In essence be the cells (or whatever is left ofthe cells when permeabilized and chemically fixed). In the typicalscenario, the change in mobility of the component is caused by thecomponent being associated with or adhered to a cellular compartment.For example, the majority of PKAcat-GFP in the inactive form aggregatesas spots in the cytoplasm. Upon activation the PKAcat spots dissolveinto the cytosol where the individual PKAcat molecules are much moremobile.

[0075] In one aspect of the invention the immobility is caused bybinding of the component to a binding partner with unknown intracellulardistribution. In this embodiment of the invention, the binding partneris fused to a protein with known substantially immobile intracellulardistribution and the component is still coupled to the luminophore.Thus, binding between the component and the binding partner will causethe component to be substantially immobile. In one aspect the change inmobility is a change from a soluble cytoplasmic state to an attachedcytoplasmic state. That is, attached to organelles, e.g. attached to IR.In another aspect the change in mobility is a change from an attachedcytoplasmic state to a soluble cytoplasmic state. In one aspect thechange in mobility is a change from a soluble cytoplasmic state to astate wherein the component is attached to the cellular membrane. Inanother aspect the change in mobility is a change from a state whereinthe component is attached to the cellular memebrane to a state whereinthe component is soluble in the cytoplasm. In one aspect the change inmobility is a change from a soluble cytoplasmic state to a state whereinthe component is attached to, or incorporated in, the nucleus. Inanother aspect the change in mobility is a change from a state whereinthe component is attached to, or incorporated in, the nucleus to asoluble cytoplasmic state. In one aspect the change in mobility is achange from an attached cytoplasmic state to a state wherein thecomponent is attached to, or incorporated in, the nucleus. In anotheraspect the change in mobility is a change from a state wherein thecomponent is attached to, or incorporated in, the nucleus to an attachedcytoplasmic state. Attachment in the cell is anticipated to be mediatedby an internal structure e.g. organelle, membrane, cytoskeleton, or amolecular structure.

[0076] It is preferred that the cellular component is taking part in anintracellular signalling pathway, such as enzymes involved in theintracellular phosphorylation and dephosphorylation processes includingkinases, protein kinases and phosphatases, but also proteins making upthe cytoskeleton play important roles in intracellular signaltransduction. In a more preferred embodiment, the cellular component isa protein kinase, a protein phosphatase, or a transcription factor.

[0077] In a preferred embodiment of the invention, the influence iscontact between the mechanically intact living cell or the group ofmechanically intact living cells with a chemical substance and/orincubation of the mechanically intact living cell or the group ofmechanically intact living cells with a chemical substance.

[0078] In one embodiment, the invention is used as a basis for ascreening program, where the effect of unknown influences such as acompound library, can be compared to influence of known referencecompounds under standardised conditions.

[0079] The extraction buffer comprises a cellular fixation agent and acellular permeabilisation agent. In one aspect of the invention thecellular fixation agent is one, or a mixture of, protein coagulants orprotein cross-linking agents, such as ethanol, acetic acid, acrolein,formaldehyde, glutaraldehyde, potassium permanganate, tannic acid,paraformaldehyde. Most preferred fixation agent is formalin. In anotheraspect of the invention the cellular permeabilisation agent is a, or amixture of, agents capable of perforating the plasma membrane of thecell such as Streptolysin O, or a anionic, non-ionic, zwitterionic orcationic detergent or detergent buffer such as lauryl sulphate,deoxycholic acid, digitonin, pluronic F68, Saponin, Triton X-100, TritonX-114, nonidet P40, CHAPS, hexadecyltrimethylammonium bromide or anagent inducing osmotic shock like salt or digitoxin. The most preferredpermeabilisation agent is Triton X-100. Any basic buffer that supportsthe cell type suitable for the experiment can be used. One preferredbasic buffer is ordinary PBS. The final wash buffer should be carefullybuffered at a pH suitable for obtaining maximal fluorescence from GFP,e.g. between pH 7.5 and 9.0, most preferably at pH 8.5. The bufferingcapacity should be sufficient to counteract the effects of trace amountsof formalin and Hoechst stain that may remain in or around cells afterwashing. The ratio between the fixation agent and the permeabilisationagent is of essential 25 importance. On the one side, the cell must bepermeabilised to let the mobile component diffuse of out the cell; onthe other side, the cell must be chemically fixed to prevent allcellular compartment to diffuse. As illustrated in Examples 10 to 12, itis of highest importance to optimized the ratio in each case since theoptimal ratio depends on the cell type and, more importantly, thecomponent and the kind of immobility (as discussed previously). Anextraction buffer comprising 0.1% Formaldehyde and 0.1% Triton-X turnsout to be a good starting point as that will, in our hands, alwaysimprove the assay even though these concentrations might not be optimal.

[0080] In order to optimize the ratio between the cellular fixationagent and a cellular permeabilisation agent, the following steps areperformed:

[0081] (a) contacting with the reference compound cells comprising aluminophore;

[0082] (b) contacting or incubating without the reference compound cellssimilar to the cells in (a);

[0083] (c) adding extraction buffer, the extraction buffer comprising acellular fixation agent and a cellular permeabilisation agent, to thecells in (a) and (b);

[0084] (d) measuring the light emitted from the luminophore;

[0085] (e) repeating steps (a) through (d) with extraction buffers withvarious concentrations of cellular fixation agent and cellularpermeabilisation agent;

[0086] (f) calculating the signal to noise (s/n) ratio as (fluorescencein stimulated cells minus the fluorescence in non-stimulated cells)divided by the fluorescence in the non-stimulated cells times 100% foreach of the extraction buffers tested in step (e); the optimizedextraction buffer being the buffer associated with the highest signal tonoise ratio. The reference compound is a compound known to cause highdegree of change in mobility of the component.

[0087] A method for decreasing background fluorescence when measuringintracellular redistribution comprising the steps of:

[0088] (a) contacting or incubating with a substance a mechanicallyintact living cell or mechanically intact living cells comprising aluminophore, the luminophore being capable of being redistributed in amanner which is related to the influence of the substance, and/or ofbeing associated with a component which is capable of beingredistributed in a manner which is related to the influence of thesubstance;

[0089] (b) adding extraction buffer, the extraction buffer comprising acellular fixation agent and a cellular permeabilisation agent to thecells; and

[0090] (c) measuring the distribution of spatially distributed light.

EXAMPLES Example 1 Construction of the Probes and Fusions

[0091] PS462 contains a fusion of PDE4A4 and EGFP under the control of aCMV promoter and has resistance to G418 as selectable marker. Toconstruct the PDE4A4-EGFP fusion, the ca. 1.9 kb C-terminal part ofHSPDE4A4 (GenBank Acc.no. L20965), which is common to all PDE4Aisoforms, is amplified using PCR with primers 4A-Ct-top and 4A-bottomdescribed below. The sequence of the top primer contains a silentmutation, which introduces a Dra1 site exactly at the beginning of theshared 4A region. The bottom primer includes the common C-terminalsequence minus the stop codon, a BamH1 cloning site, and two extranucleotides to preserve the reading frame when cloned into pEGFP-N1. Theunique ca. 0.8 kb N-terminal part of HSPDE4A4 is amplified using PCR inthe presence of 5% DMSO with primers 4A4-top and 4A4N-bottom describedbelow. The top primer includes specific HSPDE4A4 sequences following theATG, a Kozak sequence, and a Hind3 cloning site. The bottom primer spansthe junction of the unique 4A4 N-terminal part and the common 4AC-terminal part, and it contains a silent mutation that introduces aDra1 site exactly at the beginning of the shared 4A region. The PCRproducts are digested with the relevant restriction enzymes (Hind3 andDra1 for the unique N-terminal part and Dra1 and BamH1 for the commonC-terminal part), and ligated together into pEGFP-N1 (Clontech, PaloAlto; GenBank Accession number U55762) digested with Hind3 and BamH1.This produces a PDE4A4-EGFP fusion under the control of a CMV promoter.The resulting plasmid is referred to as PS462 (deposited under theBudapest Treaty with Deutsche Sammiung von Mikroorganismen undZellkulturen GmbH (DSMZ) on Apr. 17, 2000 with DSM 13450).

[0092] 4A-Ct-top: 5′- GTTTAAAAGGATGTTGMCCGTGAGCTC-3′

[0093] 4A-bottom: 5′-GTGGATCCCAGGTAGGGTCTCCACCTGA-3′

[0094] 4A4-top: 5′-GTMGCTTGCGCCATGGMCCCCCGACC-3′

[0095] 4A4N-bottom: 5′-GGTTTTAAACTTGTGCGAGGCCATCTCGCTGAC-3′

[0096] Plasmid PS642 contains a fusion of PDE4A4 and SOS1 under thecontrol of a CMV promoter, and has resistance to zeocin as selectablemarker. PS642 is derived from PS614, which is derived from PS462. PS462is constructed as described above. The neomycin resistance marker onPS462 is replaced with a zeocin resistance marker by digesting PS462with Avr2, which excises neomycin, and ligating the vector fragment witha ca 0.5 kb Avr2 fragment encoding zeocin resistance. This fragment isisolated by PCR using primers 9655 and 9658 described below with pZeoSV(Invitrogen) as template. Both primers contain Avr2 cloning sites, andflank the zeocin resistance gene including its E.coli promoter. The topprimer 9658 spans the Ase1 site at the beginning of zeocin, which can beused to determine the orientation of the Avr2 insert relative to theSV40 promoter which drives resistance in mammalian cells. The resultingplasmis is referred to as PS614.

[0097] 9658-top: TCCTAGGCTGCAGCACGTGTTGACMTTMTCATCGG-3′

[0098] 9655-bottom: TCCTAGGTCAGTCCTGCTCCTCGGCCACGMGTGCAC-3′

[0099] The coding sequence of human SOS1 (GenBank accession numberNM_(—)005633) is isolated from e.g. a human fetus or brain cDNA libraryby PCR with primers 0099 and 0100 described below. The top primerincludes specific SOS1 sequences following the ATG and a BamH1 cloningsite. The bottom primer includes specific SOS1 sequence including thestop codon and a Not1 cloning site. The PCR product is digested withrestriction enzymes BamH1 and Not1, and ligated into PS614 vector DNAdigested with BamH1 and Not1. This creates a fusion between PDE4A4 andSOS1 under the control of a CMV promoter. The resulting plasmid isreferred to as PS642.

[0100] 0099-top: 5′-GTTGGATCCCATGCAGCAGGCGCCGCAGCCTTAC -3′

[0101] 0100-bottom: 5′- GTTGCGGCCGCTCATTGGGGAGTTTCTGCATTTTC -3′

[0102] Plasmid PS587 contains a fusion of GRB2 and EGFP under thecontrol of a CMV promoter, and has neomycin resistance as selectablemarker. The coding sequence of human GRB2 (GenBank accession numberNM_(—)002086) is isolated from e.g. a human fetus or brain or placentacDNA library by PCR with primers 0073 and 0074 described below. The topprimer includes specific GRB2 sequences following the ATG and a Hind3cloning site. The bottom primer includes specific GRB2 sequenceincluding the stop codon and an EcoR1 cloning site. The PCR product isdigested with restriction enzymes Hind3 and EcoR1, and ligated intopEGFP-N1 vector DNA (Clontech, Palo Alto, GenBank Accession numberU55672) digested with Hind3 and EcoR1. This creates a fusion betweenGRB2 and EGFP under the control of a CMV promoter. The vector mayoptionally first have been modified to contain a T7 promoter (which canbe used for in vitro transcription) immediately upstream of EGFP. Thiscan be achieved by digesting pEGFP-N1 with restriction enzymes Nhe1 andBgl2, which cut Immediately upstream of EGFP, and ligating the vectorfragment with annealed oligos 9949 & 9950. The resulting plasmid isreferred to as PS587.

[0103] 0073-top: 5′-GCGMGCTTTCAGMTGGAAGCCATCG -3′

[0104] 0074-bottom: 5′-GCCGMTTCGGACGTTCCGGTTCACG -3′

[0105] 9949: 5′-CTAGCATTMTACGACTCACTATAGGGA-3′

[0106] 9950: 5′-GATCTCCCTATAGTGAGTCGTATTMTG-3′

[0107] Plasmid PS639 contains a fusion of the catalytic subunit of PKA(PKAcat) and GRB2 under the control of a CMV promoter, and hasresistance to zeocin as selectable marker. PS639 is derived from PS610,which is derived from PS457. Plasmid PS457 contains a fusion of PKAcatand EGFP under the control of a CMV promoter, and has neomycinresistance as selectable marker. The coding sequence of human PKAcat(GenBank accession number X07767) except thel7 N-terminal amino acids isisolated from e.g. a human liver or spleen cDNA library by PCR withprimers 9952 and 9922 described below. The top primer includes specificPKAcat sequences spanning an EcoR1 site near the N-terminus of thecoding sequence. The bottom primer includes specific PKAcat sequenceminus the stop codon and a BamH1 cloning site. The PCR product isdigested with restriction enzymes EcoR1 and BamH1, and ligated intopEGFP-N1 vector DNA (Clontech, Palo Alto, GenBank Accession numberU55672) digested with EcoR1 and BamH1. This intermediate is digestedwith Hind3 and EcoR1 and ligated with annealed oligos 9955 and 9956,which adds remaining N-terminal amino acids to PKAcat, and so creates afusion between PKAcat and EGFP under the control of a CMV promoter. Thisconstruct is referred to as PS457.

[0108] 9952-top: 5′GAGCGTGAAAGAATTCTTAGCCAAAG-3′

[0109] 9922-bottom: 5′-GTGGATCCCAAAACTCAGAAAACTCCTTG-3′

[0110] 9955: 5′-AGCTTCCGCGATGGGCAACGCCGCCGCCGCCAAGAAGGGCAGCGAGCAGGAGAGCGTGAAAG-3′

[0111] 9956: 5′-AATTCTTTCACGCTCTCCTGCTCGCTGCCCTTCTTGGCGGCGGCGGCGTTGCCCATC GCGGA-3′

[0112] PS610 is constructed by replacing neomycin resistance on PS457with zeocin resistance as described above. The coding sequence of humanGRB2 is isolated from PS587 described above by PCR with primers 0143 and0142 described below. The top primer includes specific GRB2 sequencesfollowing the ATG and a BamH1 cloning site. The bottom primer includesspecific GRB2 sequence including the stop codon and an Xba1 cloningsite. The PCR product is digested with restricton enzymes BamH1 andXba1, and ligated into PS610 vector DNA (isolated from a dam-minusE.coli) digested with BamH1 and Xba1. This creates a fusion betweenPKAcat and GRB2 under the control of a CMV promoter. The resultingplasmid is referred to as PS639.

[0113] 0143-top: 5′-GTTGGATCCCATGGAAGCCATCGCCAAATATG-3′

[0114] 0142-bottom: 5′-GTTTCTAGATTAGACGTTCCGGTTCACGG-3′

[0115] Plasmid PS602 contains a fusion of EGFP and the 265 C-terminalamino acids of SOS1 under the control of a CMV promoter, and theneomycin resistance marker. A C-terminal part of the coding sequence ofhuman SOS1 (GenBank accession number NM_(—)005633) is isolated from e.g.a human fetus or brain cDNA library by PCR with primers 0122 and 0123described below. The top primer includes specific SOS1 sequencesfollowing amino acid number 1067 plus an ATG and an Xho1 cloning site.The bottom primer includes specific SOS1 sequence including the stopcodon and a BamH1 cloning site. The PCR product is digested withrestriction enzymes Xho1 and BamH1, and ligated into pEGFP-C1 vector DNA(Clontech, Palo Alto, GenBank Accession number U55673) digested withXho1 and BamH1. This creates a fusion between EGFP and a C-terminal partof SOS1 under the control of a CMV promoter. The vector may optionallyfirst have been modified to contain a T7 promoter (which can be used forin vitro transcription) immediately upstream of EGFP as described above.The resulting plasmid is referred to as PS602.

[0116] 0122: 5′-GTTCTCGAGTCATGAGCTTTAGTCGGATTGCTG-3′

[0117] 0123: 5′-GTTGGATCCTCATTGGGGAGTTTCTGCATTTTC-3′

[0118] Plasmid PS628 contains a fusion of the Cys1 domain of PKCgammaand GRB2 under the control of a CMV promoter, and has resistance tozeocin as selectable marker. PS628 is derived from PS613, which isderived from PS443. Plasmid PS443 contains a fusion of the 90 N-terminalamino acids of human PKCgamma (the Cys1 domain) and EGFP under thecontrol of a CMV promoter, and has neomycin resistance as selectablemarker. The Cys1 domain of human PKCgamma (GenBank accession numbersM13977 and Z15114) is isolated from e.g. a human brain cDNA library byPCR with primers 9916 and 9935 described below. The top primer includesspecific PKCgamma sequences spanning the ATG and an EcoR1 cloning site.The bottom primer includes specific PKCgamma sequence around amino acidnumber 90 and an Acc65 cloning site. The PCR product is digested withrestriction enzymes EcoR1 and Acc65, and ligated into pEGFP-N1 vectorDNA (Clontech, Palo Alto, GenBank Accession number U55672) digested withEcoR1 and Acc65. This creates a fusion between PKCgamma-Cys1 and EGFPunder the control of a CMV promoter. This construct is referred to asPS443.

[0119] 9916: 5′-GTGAATTCGGCCATGGCTGGTC-3′

[0120] 9935: 5′-GTGGTACCTTCCCAGCGCCTGGACACTC3′

[0121] PS613 is constructed by replacing neomycin resistance on PS443with zeocin resistance as described above. The coding sequence of humanGRB2 is isolated from PS587 described above by PCR with primers 0143 and0142 described below. The top primer includes specific GRB2 sequencesfollowing the ATG and a BamH1 cloning site. The bottom primer includesspecific GRB2 sequence including the stop codon and an Xba1 cloningsite. The PCR product is digested with restriction enzymes BamH1 andXba1, and ligated into PS610 vector DNA (isolated from a dam-minusE.coli) digested with BamH1 and Xba1. This creates a fusion between Cys1and GRB2 under the control of a CMV promoter. The resulting plasmid isreferred to as PS628.

[0122] 0143-top: 5′-GTTGGATCCCATGGAAGCCATCGCCAAATATG-3′

[0123] 0142-bottom: 5′-GTTTCTAGATTAGACGTTCCGGTTCACGG-3′

[0124] To construct the HSPDE4A1-EGFP fusion, the ca. 1.95 kb codingregion of HSPDE4A1 (GenBank Acc.no. U97584) is amplified using PCR andprimers 4A1-top and 4A-boftom described below. The top primer includesspecific HSPDE4A1 sequences following the ATG, a Kozak sequence, and aHind3 cloning site. The bottom primer includes the common PDE4AC-terminal sequence minus the stop codon, a BamH1 cloning site, and twoextra nucleotides to preserve the reading frame when inserted into inpEGFP-N1. The PCR product is digested with restriction enzymes Hind3 andBamH1, and cloned into pEGFP-N1 (Clontech, Palo Alto; GenBank Accessionnumber U55762) cut with Hind3 and BamH1. This produces an HSPDE4A1-EGFPfusion under the control of the CMV promoter. The resulting plasmid isreferred to as PS461 and is deposited under the Budapest Treaty withDeutsche Sammiung von Mikroorganismen und Zellkulturen GmbH (DSMZ) onApr. 17, 2000 with DSM 13449.

[0125] 4A1-top: 5′-GTAAGCTTAAGATGCCCTTGGTGGATTTCTTC-3′, specific forPDE4A1,

[0126] 4A-bottom: 5′-GTGGATCCCAGGTAGGGTCTCCACCTGA-3′ SEQ ID NO:Description 1 DNA sequence of PDE4A4 as it appears in PS462 from startcodon to BamH1 cloning site in 3′-end. 2 Protein sequence of PDE4A4 asit appears in PS462. 3 DNA sequence of PDE4A1 as it appears in P3461,from start codon to BamH1 cloning site at 3′-end. 4 Protein sequence ofPDE4A1 as it appears in PS461. 5 DNA sequence of SOS1 as it appears inPS642, from start codon to stop codon. 6 Protein sequence of SOS1 as itappears in PS642.

Example 2 Protocols and Methods

[0127] This example describes protocols and methods used for in vivoexpression of the probes described in Example 1, and the visualisationand measurement of changes undergone by EGFP fusion probes, eithertransfected singly or as co-transfections with anchor probes in CHOcells.

[0128] Transfection and Cell Culture:

[0129] Chinese hamster ovary cells (CHO), are transfected with theplasmids described in Example 1 above, either using a single species ofplasmid, or pairs of plasmids co-transfected simultaneously, using thetransfection agent FuGENE™ 6 (Boehringer mannheim Corp, USA) accordingto the method recommended by the suppliers. Stable transfectants ofsingle GFP probes are selected using the appropriate selection agent,usually 0.5 mg/ml G418 sulphate (Calbiochem) in the growth medium (HAM'sF12 nutrient mix with Glutamax-1, 10% foetal bovine serum (FBS), 100 μgpenicillin-streptomycin mixture ml⁻¹ (GibcoBRL, supplied by LifeTechnologies, Denmark). Co-transfected cells are cultured in the samemedium but with the addition of a two selection agents appropriate tothe plasmids being used, usually 0.5 mg/ml G418 sulphate plus 1 mg/mlzeocin. Cell are cultured at 37° C. in 100% humidity and conditions ofnorrnal atmospheric gases supplemented with 5% CO₂. Clonal cell lineswith particular properties are sub cultured from mixed populations ofstably transfected cells by isolating individual cells and removing themto sterile culture flasks containing fresh culture medium with 0.5 mg/mlG418 sulphate or 0.5 mg/ml G418 sulphate+1 mg/ml zeocin as appropriateto the plasmid(s) being selected.

[0130] For fluorescence microscopy, cells are allowed to adhere toLab-Tek chambered coverglasses (Nalge Nunc International, NapervilleUSA) for at least 24 hours and are then cultured to about 80%confluence. Cells can also be grown in plastic 96-well plates(Polyfiltronics Packard 96-View Plate or Costar Black Plate, clearbottom; both types tissue culture treated) for imaging purposes. Priorto experiments, the cells are cultured over night without selectionagent(s) in HAM F-12 medium with glutamax, 100 μgpenicillin-streptomycin mixture ml⁻¹ and 10% FBS. This medium has lowautofluorescence enabling fluorescence microscopy of cells straight fromthe incubator. For certain tests requiring medium of definedcomposition, particularly with regard to the presence of specific cellgrowth factors, the HAM's culture medium is replaced prior to imagingwith a buffered saline solution (KRW buffer) containing (in mM) 3.6 KCl,140 NaCl, 2 NaHCO₃, 0.5 NaH₂PO₄, 0.5 MgSO₄, 1.5 CaCl₂, 10 Hepes, 5glucose, pH7.4.

[0131] Confocal Imaging:

[0132] Confocal images are collected using a Zeiss LSM 410 microscope(Carl Zeiss, Jena, Germany) equipped with an argon ion laser emittingexcitation light at 488 nm. In the light path are a FT510 dichroicbeamsplitter and a 515 nm long-pass filter or a 510 to 525 nm bandpassemission filter. Images are typically collected with a Fluar 40X, NA:1.3 oil immersion objective, the microscope's confocal aperture set to avalue of 10 units (optimum for this lens).

[0133] Time Lapse Sequences and Analysis:

[0134] Image sequences of live cells over time (time lapse, e.g. FIGS.5, 6, 11, 12) are gathered using a Zeiss Axiovert 135M fluorescencemicroscope fitted with a Fluar 40X, NA: 1.3 oil immersion objective andcoupled to a Photometrics CH250 charged coupled device (CCD) camera(Photometrics, Tucson, Ariz. U.S.A.). The cells are illuminated with a100 W HBO arc lamp. In the light path are a 470±90 nm excitation filter,a 510 nm dichroic mirror and a 515±15 nm emission filter for minimalimage background. The cells are maintained at 37° C. with a custom-builtstage heater. Time lapse response profiles are extracted from imagesequences using a region of interest (ROI) defined over the sameco-ordinates or pixels for each successive image in a sequence: pixelvalues are summed and averaged over the ROI in each image, and theresulting values plotted against image number to generate a time lapseresponse profile for that defined region of the sequence. A ROI caninclude many cells, a single cell, or a region within a single cell.

[0135] Automated Imaging and Analysis:

[0136] The amount of fluorescent spots and other accumulations of atransfected probe in a population of cells can be imaged and quantifiedin an automated fashion to yield a measure of mean number of spots percell. For this purpose cells are cultured to near 80% to 90% confluencein coverglass chambers or plastic 96-well plates, given the relevanttreatment and allowed to respond. At the end of the response period,cells are chemically fixed in 4% formaldehyde buffer (Lillies fixativebuffer, pH7.4: Bie and Berntsen A/S, Denmark) for 30 minutes to 2 hours,and then washed three times in phosphate buffered saline (PBS, LifeTechnologies, Denmark). An alternative simultaneousfixation+permeabilisation method, useful to remove non-localised (i.e.mobile) GFP probe from the cytoplasm, involves a single fixation processincorporating 0.4% to 2% formaldehyde buffer (10% to 50% strengthLillies fixative) plus 0.2% to 1% Triton X-100. The actualconcentrations used need to be optimised for the cell type being used;for CHO cells 2% formaldehyde +1% Triton X-100 gives excellent results.The combined fixative+detergent are applied to the cells for 10 to 20minutes at room temperature. Cells are then washed three times withphosphate buffered saline. Nuclear DNA is stained with 10 μM Hoechst33258 (Molecular Probes, Eugene, Oreg., U.S.A.) in PBS for 10 minutes at25° C., then washed twice in PBS. Automated images are collected on aNikon Diaphot 300(Nikon, Japan) using a Nikon Plan Fluor 20X/0.5NAobjective lens. The basic microscope is fifted with a motorised specimenstage and motorised focus control (Prior Scientific, Fulbourn, CambridgeUK), excitation filter wheel (Sutter Instruments, Novato Calif. U.S.A.)and Photometrics PXL series camera with a KAF1400 CCD chip(Photometrics, Tucson, Ariz. U.S.A.), each of these items being underthe control of a Macintosh 7200/90 computer (Apple Computer, Cupertino,Calif. U.S.A.). Automation of stage positioning, focus, excitationfilter selection, and image acquisition is performed using macroswritten in-house, running under IPLab Spectrum for Macintosh(Scanalytics, Fairfax, Va. U.S.A.). Fluorescence illumination comes froma 100 W HBO lamp. Images are collected in pairs, the first using a340/10 nm excitation filter, the second with a 475RDF40 excitationfilter (Chroma, Brattleboro, Vt.). Both images are collected via thesame dichroic and emission filters, which are optimised for EGFPapplications (XF100 filter set, Omega Optical, Brattleboro, Vt.). Whilethe choice of filters for imaging the nuclear stain (Hoechst 33258) isnot well matched to that dye's spectral properties, resulting in lowerimage intensity, it greatly improves the throughput of the procedure byallowing both images to be collected using the same dichroic andemission filter. This eliminates any image registration problems andfocus shifts which would result from using two different filter sets,which would require more steps in the acquisition procedure and moreextensive image processing to overcome.

[0137] The necessary images are collected as follows: A holdercontaining four 8-well coverglass chambers, or a single 96-well plate,is loaded onto the microscope. The program is started, and the firstwell of cells is moved into position and manually coarse-focused by theoperator. The image is fine-focused by an auto-focus routine using the340/10 excitation. An image is captured and stored at this excitationwavelength (the nuclear image), and then a second image is captured andstored at the longer wavelength excitation (the GFP image). The stage isautomatically repositioned and microscope automatically refocused tocapture a second pair of images within the same well. This process isrepeated a set number of times (typically 4 to 8) for the first well.The stage then advances the next well to the imaging position, and theprocess repeats itself until the set number of image pairs has beencaptured from each well of cells.

[0138] Image pairs are automatically analysed in the following way usinga suite of macros running under the IPLab Spectrum software: First thenuclear image of a pair is filtered with a digital filter tosimultaneously sharpen the edges of and suppress differences inintensity of the nuclei. The choice of filter, and the filter constants,were arrived at through experimentation with various data sets. Thefiltered image is then segmented at a pre-determined intensity value,such that pixels below this threshold are very likely not within anuclear region, and pixels above this threshold are very likely within anuclear region. The contiguous regions above the threshold are thencounted, after rejecting contiguous regions that are larger than acertain area or smaller than a certain (different) area, the areashaving been previously determined to provide a sufficiently accuratedistinction between nuclei and other objects that are not nuclei. Thefinal count is the estimated number of nuclei in the field. The GFPimage of each pair is then digitally filtered with a filter chosenexperimentally to suppress the variation of intensity due to the typicalnon-localised distribution of GFP, while accentuating the intensity ofany bright point-like objects relative to this background. This filteredimage is then segmented at a threshold that has been experimentallydetermined to divide the image into pixels that are very likely to be ina spot (above the threshold) and pixels that are very likely not to bein a spot (below the threshold). The contiguous regions of pixels thatare above the threshold are counted, after rejecting regions that do nothave certain morphological properties, which were previously determinedto be characteristic of spots. The ratio of spot count to nuclear countfor each pair represents an estimate of the average number of spots percell in that image pair. All image pairs are treated in this way, andthe final table of values is used to establish the cellular response toa given treatment. FIG. 3 is an example of how the effect of a compoundupon spot density in CHO cells expressing HSPDE4A4-EGFP can be measuredusing the automated imaging and image analysis method. The amount offluorescent spots and other accumulations of a transfected probe in apopulation of cells can also be quantified using a fluorescent platereader, such as a TECAN Spectrafluor (Tecan U.S. Inc., Research TrianglePark, N.C. 27709, U.S.A.) or using a fluorescence imaging plate reader(FLIPR; Molecular Devices Corp., Sunnyvale, Calif. 94089, U.S.A.). Theuse of the combined fixation+permeabilisation method greatly improvesthe signal over background for measurements of spots and otheraccumulations in cells, particularly when the measurements are made onfluorescent plate readers. FIG. 4 is an example of the use of the FLIPRto measure the effect of a compound upon spot density in CHO cellsexpressing HSPDE4A4-EGFP. The cells were treated with the simultaneousfix and permeabilisation protocol to obtain these data. Redistributionsof fluorescent probes from cytoplasm to plasma membrane may bequantified by standard imaging methods using simple image analysis ofthe changes in fluorescence intensity of cytoplasmic ROls. Similarredistributions may also be measured on the FLIPR, and even on standardfluorescent plate readers, especially those configured to measure signalfrom adherent cells in microtitre plates. Use of the FLIPR to measurePKC-like redistributions (of which EGFP-Cysl(PKCy) is an example), andalso PKAc-like redistributions, in real time, is detailed in an earlierpatent AN IMPROVED METHOD FOR EXTRACTING QUANTITATIVE INFORMATIONRELATING TO AN INFLUENCE ON A CELLULAR RESPONSE (WO 00/23615). FIG. 7shows how the effect of a compound on the redistribution of Cys1γ-EGFPcan be quantified using the FLIPR.

Example 3 Probes for Proteins X and Y with PDE4A4 Anchor Protein

[0139] The present example and Example 4, Example 5 and Example 6,describe generic ways to produce a cell line suitable for screeningcompounds targeting against a specific interaction between two partnercomponents X and Y. In these examples the cells are derived from CHOcells co-transfected with two plasmids, one coding for fusion probeswith X or Y attached to either the C or N terminal of the anchor moiety(the anchor probe), and the second with the other partner, Y or X,attached to either the C or N terminal of GFP (the detectable probe).There are therefore 4 possible anchor probes and 4 possible detectableprobes that can be made for the interacting pair X-Y, and 8 usefulcombinations to co-transfect. Anchor and detectable probes use differentselection markers to ensure that cells under selection maintain bothplasmids; for example, the anchor may confer resistance to zeocin, thedetectable to neomycin. Cells that maintain both probes under continuousselection (minimum of 2 weeks) are termed “stable”. In this example theanchor probes are based on HSPDE4A4B (human phosphodiesterase type 4,isoform A, splice variant 4B: referred to hereafter as PDE4A4), whichwhen treated with the PDE4 inhibitor rolipram forms a number of denseaggregates in the cytoplasm of each cell (EC₅₀ 0.2 to 0.3 μM). Theserolipram-induced aggregates may be detected with an antibody directedagainst the unique C-terminal peptide sequence of HSPDE4A, asdemonstrated in FIG. 10. In cells successfully transfected with aplasmid coding for a fusion protein having GFP fused to the C-terminalof PDE4A4, the fluorescence is distributed in a general and fairly evenway within the cytoplasm (FIG. 1), but when treated with rolipramaggregates form and these are apparent as bright green fluorescent spotsin the cell (FIG. 2). There are on average 2 such aggregates perrolipram-treated cell positioned diametrically opposite each othereither side of the nucleus. After 10 to 16 hours of exposure to 10 μMrolipram these aggregates grow to about 2 to 4 μm in diameter. Removalof rolipram causes these aggregates to disperse within 60 minutes.Similar spots appear in PDE4A4-transfected cells if treated with thePDE4 inhibitors RS25344 (EC₅₀ 0.02 μM, Syntex) and Ro 20-1724 (EC₅₀ 4μM, Roche). In cells co-transfected with PDE4A4-anchor probe-X plusEGFP-Y detectable probe, GFP-bright spots will only appear in cellsafter treatment with rolipram if X and Y interact and attract each other(FIG. 8). The spots will disappear (FIG. 9) when rolipram is removed, orcompeted against with RP73401 (Piclamilast, Rhone-Poulenc Rorer) anotherpotent PDE4 inhibitor (IC₅₀ versus 3 μM rolipram approx. 20 nM, FIGS. 3and 4).

[0140] Protocol for PDE4A4 Anchor System:

[0141] 1) Co-transfect a PDE4A4 anchor-detectable pair into CHO

[0142] 2) Check there are no GFP-bright spots to start. If there arespots, or other distinct distribution of GFP present, may still be worthtesting cells at (3): a change in distribution must be seen in step (3)if this transfection is to be useful.

[0143] 3) Test transients with 10 μM rolipram and (separately) with 1 μMRS25344, overnight

[0144] a. spots appear: clone cells from transients and put under doubleselection (as in 4). Cells ready for screening when stable.

[0145] b. no spots: wait for stable cell line to form from cells underdouble selection (see 4)

[0146] 4) Make stable cell line using double selection (e.g. 1 mg/ml ofzeocin plus 0.5 mg/ml neomycin/G418). Test separately with 10 μMrolipram and 1 μM RS25344

[0147] a. spots appear: clone cells from stables under double selectionready for screening use.

[0148] b. no spots: fix and stain (rolipram or RS25344-treated cells)with anti-4A antibody

[0149] i. spots stain with antibody: go to (5)

[0150] ii. no spots: anchor system not present or not working—repeat theco-tranfection or try another anchor system or orientation.

[0151] 5) Test for conditional association (see A and B below).

[0152] 6) If no association conditions are found, choose anothercombination of anchor/detectable pairs and start again.

[0153] Test for conditional association with an appropriate interactionstimulus: A and B can be done in parallel. B tests for possibleconditions or associations that can only occur in the “soluble phase”.

[0154] A i) incubate stables overnight with 10 M rolipram and separatelywith 1 M RS25344

[0155] ii) test with an interaction stimulus, i.e. a treatment likely tobring pair together. NB this may need time-lapse to catch transients:

[0156] a. spots appear clone cells from stables

[0157] b. No spots: try (B)

[0158] B i) incubate stables overnight with 10 μM rolipram andseparately with 1 μM RS25344

[0159] ii) washout rolipram or RS, incubate 60 minutes (to allow PDE4A4anchor to be liberated from its attachment)

[0160] iii) test with an interaction stimulus, i.e. a treatment likelyto bring pair together, together with (or followed quickly by) 10 μMrolipram or 1 μM RS25344, as appropriate:

[0161] a. spots appear clone cells from stables

[0162] b. No spots: see (6)

Example 4 Probes for Proteins X and Y with PKAcatα Anchor protein

[0163] In this example the anchor probes are based on HSPKAcatα(catalytic subunit of human cyclic AMP-dependant protein kinase, isoformα: hereafter referred to as PKAc), which when transfected into cellsforms aggregates in the cytoplasm under conditions of low cytoplasmiccAMP concentrations. These aggregates may be detected with an antibodydirected against PKAc, also with antibodies directed against the PKAregulatory subunit type 1α (R1α). In cells successfully transfected witha plasmid coding for a fusion protein having GFP fused to the C-terminalof PKAc, such aggregates are seen as bright green fluorescent spots inthe cell (FIG. 5). These aggregates disperse into the cytoplasm whencAMP is elevated in the cell, for example when cells are treated with 25μM forskolin plus 500 μM isobutyl methyl xanthine (IBMX). In cellsco-transfected with PKAc anchor probe plus EGFP detectable (X on one, Yon the other), GFP-bright spots will only appear in cells if X and Yinteract and attract each other (FIG. 11a). The spots will disappearwhen cAMP is elevated in the cells (FIG. 11b).

[0164] Protocol for PKAc Anchor System:

[0165] 1) Co-transfect an anchor-detectable pair into CHO

[0166] 2) Check distribution in transients

[0167] a) spots present: clone cells from transients and put underdouble selection (as in 3). Cells ready for screening when stable.

[0168] b) no spots: wait for stables (3)

[0169] c) If other distribution present, distinct from normal PKA spots,test with 50 μM forskolin +500 μM IBMX.

[0170] i) if distribution changes, clone cells ready for use inscreening under double selection as described in (3).

[0171] ii) No change: may still be worth continuing to stables at (3)

[0172] 3) Make stable cell line using double selection (e.g. 1 mg/ml ofzeocin plus 0.5 mg/ml neomycin/G418).

[0173] a) spots appear: test with 50 μM forskolin+500 μM IBMX; ifdistribution changes, clone cells under double selection ready for usein screening.

[0174] b) no spots: fix and stain with anti-PKAcat antibody

[0175] i) spots stain with PKAcat antibody: go to (4)

[0176] ii) no spots: anchor system not present or not working, or cAMPtoo high stain with antibody to PKA regulatory subunit type 1α(R1α)—this will reveal PKA “spots” even if catalytic subunit detached(high cAMP): if no spots seen, retransfect.

[0177] 4) Test for conditional association (see A and B below).

[0178] 5) If no association conditions are found, choose anothercombination of anchor/detectable pairs and start again.

[0179] Test for conditional association with an appropriate interactionstimulus: A and B can be done in parallel. B tests for possibleconditions or associations that can only occur in the “soluble phase”.

[0180] A i) test with an interaction stimulus, i.e. a treatment likelyto bring pair together. NB this may need Ume-lapse to catch transients:

[0181] (a) spots appear: clone cells ready for use in screening.

[0182] (b) No spots: try (B)

[0183] B i) incubate stables with 50 μM forskolin+500 μM IBMX for 30minutes (to allow PKA anchor to be liberated)

[0184] ii) washout forskolin+IBMX, test with an interaction stimulus,i.e. a treatment likely to bring pair together, incubate for 30 to 60minutes:

[0185] (a) spots appear: clone cells ready for use in screening.

[0186] (b) No spots: see (5).

Example 5 Probes for Proteins X and Y with PDE4A1 Domain Anchor Protein

[0187] In this example the anchor probes are based on PDE4A1 domains.PDE4A1 accumulates as small perinuclear spots in otherwise untreatedcells. These spots are readily detected wioth an antibody raised againstthe unique C-terminal portion of PDE4A. Treatment with rolipram causesthese spots to disperse into the cytoplasm (FIG. 13). Subsequent removalof rolipram results in the rapid re-appearance of perinuclear spots.

[0188] In FIG. 13a CHO cells stably expressing HSPDE4A1-GFP are growingin only HAM's F12 medium with 10% FBS; the GFP fluorescence isrestricted to bright granule-like spots within the perinuclear cytoplasmof each cell. The spots may be clustered around, in or on the Golgimembranes. In FIG. 13b similar cells to those seen in 13 a have beentreated with 2 micromolar rolipram for 2 hours. The majority ofGFP-bright spots disappear in all cells under rolipram treatment, andthe cytoplasm becomes generally brighter. Larger spots may not dispersecompletely in some cells. When rolipram is washed away, the spots reformwithin 1.75 hours. Furthermore, the distribution of PDE4A1, and anychange thereof, is readily measurable by automated imaging. FIG. 14shows a dose response curve for spot dispersal in response to rolipram.The number of spots per cell for each concentration of rolipram is themean of 4 measurements±sem, where each measurement is itself an averagetaken from not less than 100 cells. Cells are grown in HAM's F12 mediumplus 10% FBS plus various concentrations of rolipram for 7 hours. Thecells are then chemically fixed with 4% formalin buffer (pH7.5) for 15minutes, washed with PBS and stained with 10 PM Hoechst 33258 in PBS for10 minutes at 25° C., then washed twice in PBS. Automated images arecollected and analysed for the number of spots per cell as described inExample 2.

[0189] Protocol for PDE4A1 Anchor System:

[0190] 1) Co-transfect an anchor-detectable pair into CHO

[0191] 2) Check distribution in transients

[0192] a) spots present: clone cells from transients and put underdouble selection (as in 3).

[0193] Cells ready for screening when stable.

[0194] b) no spots: wait for stables (3)

[0195] c) If other distribution present, distinct from normal PDE4A1spots, test with 10 μM rolipram.

[0196] i) if distribution changes, clone cells ready for use inscreening under double selection as described in (3).

[0197] ii) No change: may still be worth continuing to stables at (3)

[0198] 3) Make stable cell line using double selection (e.g. 1 mg/ml ofzeocin plus 0.5 mg/ml neomycin/G418).

[0199] a) spots appear test with 10 μM rolipram; if distributionchanges, clone cells under double selection ready for use in screening.

[0200] b) no spots: fix and stain with anti-PDE4A antibody

[0201] i) spots stain with PDE4A antibody: go to (4)

[0202] ii) no spots: anchor system not present or not working;retransfect.

[0203] 4) Test for conditional association (see A and B below).

[0204] 5) If no association conditions are found, choose anothercombination of anchor/detectable pairs and start again.

[0205] Test for conditional association with an appropriate interactionstimulus: A and B can be done in parallel. B tests for possibleconditions or associations that can only occur in the “soluble phase”.

[0206] A ii) test with an interaction stimulus, i.e. a treatment likelyto bring pair together. NB this may need time-lapse to catch transients:

[0207] (c) spots appear clone cells ready for use in screening.

[0208] (d) No spots: try (B)

[0209] B i) incubate stables with 10 μM rolipram for 7 hours (to allowPDE4A1 anchor to be liberated)

[0210] ii) washout rolipram, test with an interaction stimulus, i.e. atreatment likely to bring pair together, incubate for 2 to 4 hours:

[0211] (c) spots appear: clone cells ready for use in screening.

[0212] (d) No spots: see (5).

Example 6 Probes for Proteins X and Y with Cys1 Domain Anchor Protein

[0213] In this example the anchor probes are based on the Cys1 domain ofPKCγ (Cys1 domain of protein kinase C isoform γ) in which codingsequences for the myc or flag antigens are optionally included, whichwhen transfected into cells has a general cytoplasmic distribution thatredistributes to the plasma membrane when treated with PMA(phorbol-12-myristate-13-acetate). The redistribution from cytoplasm toplasma membrane may be detected with an antibody directed against theCys1 domain of PKCγ, or with antibodies directed against the myc or flagantigens, if these have been engineered into the Cys1 construct. Incells successfully transfected with a plasmid coding for a fusionprotein having GFP fused to the C-terminal of the Cys1 domain of PKCγ,with or without the myc or flag antigen sequences, the PMA-inducedredistribution is observable as a decrease in GFP fluorescence in thecytoplasm with a concomitant increase in GFP fluorescence at the plasmamembrane (FIG. 6). In cells co-transfected with Cys1 domain of PKCγanchor probe plus EGFP detectable probe (X on one, Y on the other), GFPfluorescence will only redistribute to the plasma membrane upon PMAtreatment if X and Y interact and attract each other (FIG. 12).

[0214] Cys1 Anchor.

[0215] 1) Co-transfect new anchor-detectable pair into CHO

[0216] 2) Check that distribution is cytoplasmic in transients. Ifspots, or other distinct distribution present, may still be worthtesting at (3), but only if a change in distribution is be measurable.

[0217] 3) Test transients with 100 nM PMA for 30 minutes

[0218] a) Probe redistributes to plasma membrane: clone cells fromtransients under double selection as in (4)

[0219] b) no redistribution: wait for stables (see 4)

[0220] 4) Make stable cell line using double selection (e.g. 1 mg/ml ofzeocin plus 0.5 mg/ml neomycin/G418). Test with PMA

[0221] a) Probe redistributes to plasma membrane: under double selectionready for use in screening.

[0222] b) no redistribution: fix and stain with anti-myc antibody

[0223] i) PM stains with antibody: go to (5)

[0224] ii) no PM stain: anchor system not present or notworking—retransfect.

[0225] 5) Test for conditional association (see A and B below).

[0226] 6) If no association conditions are found, choose anothercombination of anchor/detectable pairs and start again.

[0227] Test for conditional association with an appropriate interactionstimulus: A and B can be done in parallel. B tests for possibleconditions or associations that can only occur in the “soluble phase”,before targeting to PM is imposed.

[0228] A i) incubate stables with 100 nM PMA for 30 minutes

[0229] ii) test with an interaction stimulus, i.e. a treatment likely tobring pair together. NB this may need time-lapse to catch transients:

[0230] (a) redistribution to PM occurs: clone cells from stables etc.

[0231] (b) No redistribution: try (B)

[0232] B i) test with an interaction stimulus, i.e. a treatment likelyto bring pair together, together with (or followed quickly by) 100 nMPMA

[0233] (a) redistribution to PM occurs: clone cells from stables underdouble selection as in (4)

[0234] (b) No redistribution: see (6)

Example 7 Assessment of Sos and Grb2 Interactions with PDE4A4

[0235] This example demonstrates the use of the redistribution trapmethod to create a cell line suitable for screening compounds againstthe interaction between Sos and Grb2, components involved in thesignalling pathway immediately downstream from certain tyrosine kinasereceptors located at the plasma membrane of mammalian cells, for examplethe insulin and the epidermal growth factor receptors. Sos is a guaninenucleotide exchange factor responsible for activation Ras-like GTPases,Grb2 an adaptor component responsible for recuiting Sos to the activatedreceptor. In this example the anchor probe is based on HSPDE4A4. CHOcells are stably co-transfected with anchor probe HSPDE4A4-SosA and“detectable” probe hGrb2-EGFP as described in Example 2 and Example 3.The anchor probe is selected for with zeocin, and the detectable probewith neomycin (G418). FIG. 8a is a confocal image of such cells growingin HAM's F12 medium with 10% FBS and FIG. 8b shows similar cells aftertreatment with 10 μM rolipram. The transfected cells are a mixednon-clonal population. The GFP-labelled detectable probe (GFPfluorescence) is distributed throughout the cell prior to rolipramtreatment, with a slightly higher concentration in the nuclei. Afterrolipram treatment (20 hours) some cells, in clonal groups, developdistinct bright spots (FIG. 8b). The appearance of the spots, and therequirement for rolipram, indicate that in these cells the anchor probehas responded to rolipram as PDE4A4 is expected to (FIG. 2), and thatthe GFP-labelled detectable probe must be associated with it. To confirmthat the spots are the result of a rolipram induced effect on the anchorprobe, the PDE4 specific inhibitor RP73401 is applied in the presence ofrolipram (FIG. 9). RP73401 competes with rolipram and causes PDE4A4spots to disperse (FIG. 3 and 4). As can be seen in FIG. 9, the spotsdisappear with RP73401 treatment, confirming that it is the anchor probethat is responsible for the spotty distribution of the detectable probe,and thus that anchor and detectable pairs are interacting. SinceHSPDE4A4 and EGFP do not interact when co-transfected and rolipramtreated (not shown, but EGFP distribution is always pan-cellular, inboth nucleus and cytoplasm in such cells, and its distribution isunaffected by rolipram), the interaction must be mediated by the naturalinteraction of Sos and Grb2. When cloned, the co-transfected cells maybe used for screening compounds against the Sos-Grb2 interaction.Compounds, which make the rolipram-induced spots, disappear in theseco-transfected cells, but which do not affect rolipram-induced spots incells stably expressing only the HSPDE4A4-EGFP probe, are compounds,which specifically disrupt the interaction between Sos and Grb2.

Example 8 Assessment of Sos and Grb2 Interactions with PKAcatα

[0236] This example describes the creation of a cell line suitable forscreening compounds against the interaction between Grb2 and theC-terminal sequence of Sos (amino acids 1067 to 1332 of Sos). This issimilar to the interaction pair described in Example 7, except that theanchor probe is based upon the catalytic subunit of cAMP-dependentprotein kinase isoform α (HSPKAcat-hGrb2), and therefore locates inunstimulated cells as aggregates, which if they attract the detectableprobe (EGFP-Sos-Cterm) will become GFP-bright. One potential advantageof this anchor system is that a prior anchor stimulus is not needed inorder to appreciate an interaction, but is used as a confirmatory testafter the interaction has been identified. The process of screening forinteraction modulators with this system therefore involves no likelihoodof changing the activity of other cellular signaling pathways which mayinterfere with the interaction under test. Treatment with forskolin±IBMX(isobutyl methylxanthine, a non-specific PDE inhibitor) is used in thePKAcat system to confirm that any GFP-bright aggregates are the resultof a specific interaction between anchor and detectable probes in thissystem, since aggregates of PKAcat will dissolve into the cytoplasmunder this treatment. CHO cells are stably co-transfected with theanchor probe HSPKAcat-hGrb2 and GFP-labelled detectable probeEGFP-Sos-Cterm as described in Example 2 and Example 4. The anchor probeis selected for with zeocin, and the detectable probe with neomycin(G418). CHO cells stably expressing both the anchor probe HSPKAcat-hGrb2and GFP-labelled detectable probe EGFP-Sos-Cterm are shown in FIG. 11before (FIG. 11a) and 10 minutes after (FIG. 11b) treatment with 50micromolar forskolin+500 micromolar IBMX. Three cells are evident in(FIG. 11a) with GFP-bright aggregates typical of the distribution ofPKAcat-EGFP seen in unstimulated cells (FIG. 5, t=0 minutes). Thedispersal of these spots by forskolin+IBMX, a treatment which increasesCAMP in the cells, shows that the GFP-labelled detectable probe must beinteracting with the anchor probe in the co-transfected cells, becauseonly the anchor probe can react to increased cAMP in this way (FIG. 5).Cells showing spots are suitable for cloning and may then be used forscreening compounds against the interaction between Grb2 and Sos-Cterm.Compounds which make the GFP-bright aggregates disappear in theseco-transfected cells, but which do not affect aggregates in cells stablytransfected with PKAcat-EGFP probe alone, are compounds whichspecifically disrupt the interaction between Sos-Ctern and Grb2.

Example 9 Assessment of Sos and Grb2 Interactions with Cys 1 Domain

[0237] This example describes the creation of another cell line suitablefor screening compounds against the interaction between Grb2 and theC-terminal sequence of Sos (amino acids 1067 to 1332 of Sos). This isthe same interaction pair described in Example 6, except that the anchorprobe is based upon the Cys1 domain of protein kinase C isoform gamma,and therefore locates in unstimulated cells throughout the cytoplasm andnucleus. When these cells are treated with 100 nM PMA the anchor proberedistributes to the plasma membrane, and if an interaction occursbetween anchor and detectable probes, the GFP fluorescence will also beseen to redistribute to the plasma membrane. An advantage of this anchorsystem is that when stimulated with PMA, the anchor probe is taken tothe plasma membrane where it will come into close proximity with othercomponents that may be important in modulating the interaction betweenthe components attached to the anchor and EGFP moieties. In the case ofGrb2 and Sos-Cterm proximity to the plasma membrane does not appear tobe necessary in order to initiate their interaction, but certaincomponent pairs may fail to interact unless this condition is met.Treatment with PMA is used to confirm that an interaction between anchorand detectable probes occurs in this system, since redistribution offluorescence to the plasma membrane can only occur if interaction of thetwo probes has first taken place. This can be checked in CHO cellstransfected with EGFP-Sos-Cterm alone, where PMA has no effect on thedistribution of fluorescence. CHO cells are stably co-transfected withthe anchor probe Cys1-hGrb2 and the GFP-labelled detectable probeEGFP-Sos-Cterm as described in Example 2 and Example 6. The anchor probeis selected for with zeocin, and the detectable probe with neomycin(G418). FIG. 12 shows CHO cells stably expressing both the anchor probeCys1-hGrb2 and GFP-labelled detectable probe EGFP-Sos-Cterm before (FIG.12a) and 5 minutes after (FIG. 12b) treatment with 100 nM PMA. Themajority of cells in before PMA treatment have a rather generalcytoplasmic and nuclear distribution of fluorescence. Treatment with PMAcauses a measurable shift in fluorescence to the plasma membrane, in thesame way that Cys1-EGFP would redistribute in response to PMA (FIG. 6).This result shows that the GFP-labelled detectable probe must beinteracting with the anchor probe, because only the anchor probe canreact to PMA in this way. Cells showing this behaviour are suitable forcloning and may then be used for screening compounds against theinteraction between Grb2 and Sos-Cterm. Compounds which preventPMA-induced redistribution of EGFP fluorescence in these co-transfectedcells, but which do not affect fluorescence redistribution in cellsstably transfected with the Cys1-EGFP probe alone, are compounds, whichspecifically disrupt the interaction between Sos-Cterm and Grb2.

Example 10 Assay Procedure for Trap Assay

[0238] The selective PDE4 inhibitor rolipram affects the physicalproperties and behaviour of PDE4A4 such that the general cytoplasmicdistribution of PDE4A4 in most cells gradually changes to one consistingof concentrations of PDE4A4 located at several distinct spots within thecytoplasm (see Example 3). This example illustrates how binding betweenthe 14-3-3 protein and BAD can be visualized using the PDE4A4-14-3-3betaand eGFP-BAD fusions. It is an example of a change in mobility from asoluble cytoplasmic state to a state wherein the eGFP-BAD fusion isattached, or actually aggregated in the cytoplasm.

[0239] Chemicals used:

[0240] NUT.MIX F-12(HAM) with GLUTAMAX-1. Gibco Brl 31765-027.

[0241] “FCS”, Foetal Bovine Serum, Gibco Brl 10084-168.

[0242] Pen/strep (100 u/ml / 100 μg/ml, Gibco cat Brl 15140-122)Formaldehyde 4%. Bie & Bemtsen Lab 00220.

[0243] Triton-x 100, Sigma Hoechst 33258 (stock solution: 10 mM)rolipram, RBI DMSO, Sigma D-5879.

[0244] Instrumentation:

[0245] Fluoscan ASCENT CF plate reader (Labsystems)

[0246] Filters:

[0247] 485/527 (GFP) and 355/460 (Hoechst) all from Labsystems

[0248] Buffers:

[0249] Extraction buffer: Formaldehyde 4% diluted 1:40 (=0.1%) inPBS+Triton-x 100 1%+Hoechst33258 10 μM. PBS Dulbecco's: GIBCO 14190-094

[0250] Cells:

[0251] Cell type: CHO cells transfected with one plasmid expressing anin frame protein fusion between PDE4A4 and human 14-3-3beta and anotherplasmid expressing an in frame protein fusion between EGFP and human BAD(details on the construction of such probes can be found in examples 1and 3). Density: 1.0×10E5/well (the day they are seeded), in 96-wellmicrotitre plates (ViewPlate-96, Black; Packard)

[0252] Procedure:

[0253] Cells are plated in 1.0×10E5/well in 200 μl HAM F-12 w. 10% FCSand 5% pen/strep 20-24 hrs before screening. Stimulated cells arefurther added 10 μM rolipram. For testing drugs capable of inhibitingthe interaction between 14-3-3 and BAD the test compound is added on topof the 200 μl HAM F-12 growth medium and incubated for 2 hrs in CO₂incubator HAM F-12 with test compound is drained from all wells of themicrotitre plate and cells are extracted in 200 μl extraction buffer for5 min. Cells are added 100μl formaldehyde 4% buffer on top of the 200μlextraction buffer and are incubate for 5 min. Extraction buffer isdrained and cells are washed×2 in 100 μl PBS. Cells are added 200 μl PBSbuffer. The fluorescence is read in ASCENT plate reader using apre-programmed procedure and filter settings suitable for GFP andHoechst 33258 fluorophores. The Hoechst reading is used to provide asignal by which cell number can be estimated, and thereby useful innormalising GFP signals from wells that may not contain identical numberof cells. The results are presented in FIG. 15.

Example 11 Assay Procedure for Redistribution™ Assay

[0254] It is well established that NFkB(p65) moves from cytosol tonucleus upon stimulation of NFkB with II-1. This can be visualized witha NFkB(p65)-GFP fusion. The total GFP intensity measured after removingcytosolic NFkB(p65)-YGFP is indicative of the degree of translocation tothe nucleus, and thus of the degree of activation of NFkB. This is anexample of a change in mobility as a change from a soluble cytoplasmicNFkB to a component attached in the nucleus. Applying the extractionprocedure, the difference between non-stimulated and stimulated cellscan be read by the intensity of fluorescence (see FIG. 16).

Example 12 Extraction Optimisation

[0255] Cells are seeded in a known concentration and grown for 24 hrs.Stimulated and non-stimulated cells (representing the full “dynamic”range) are extracted with different combinations of triton-x 100 andformalin and the signal is read in a plate reader. The aim is toidentify the ratio between formalin and triton-x that gives the optimalsignal to noise (s/n) ratio. The s/n is calculated as (fluorescence instimulated cells minus the fluorescence in non-stimulated cells) dividedby the fluorescence in the non-stimulated cells times 100%.${s/n} = {\frac{\left( {{{stimulated}\quad {cell}} - {{non}\quad {stimulated}\quad {cells}}} \right)}{{non}\quad {stimulated}\quad {cells}}*100\quad \%}$

[0256] Optimisation of the extraction buffer for binding between the14-3-3 protein and BAD

[0257] The optimization is illustrated in FIG. 17.

Example 13 Quantification of Forskolin Induced PKAcat Translocationusing Extraction

[0258] PKAcat-GFP forms distinct spots in its inactive tetrameric form.Upon increase in cellular cAMP (e.g. by stimulation of adenylate cyclaseactivity by forskolin) PKAcat dissociates from its regulatory subunitsand spots “dissolves” into the cytosol. The total GFP intensity measuredafter removing cytosolic PKAcat-GFP through the extraction procedure isindicative of the level of non-activated, non-dissociated PKAcat. Inthis example the extraction procedure is used to quantify a forskolindose response in a PKAcat-GFP assay. Cells are treated with forskolin invarious conc. for 15 min. (remove spots). Cells are extracted (Formalin0.2%/Triton-x 0.2%). Signal is read in FLIPR. Results presented in FIG.18. The determined ED50 at about 3 μM is in accordance with literaturestandards.

Example 14 Comparison of the Effect of Rolipram Treatment upon NormalCHO Cells and upon CHO Cells Stably Expressing a HSPDE4A4B-GFP FusionProtein

[0259] The PDE4 inhibitor rolipram is used to stimulate the productionof the dense aggregates 5 of PDE4A4 in the cytoplasm of cellstransfected with fusions to PDE4A4 described in Example 3. A possibleconcern of using such a stimulus is that levels of the second messengermolecule cAMP, the substrate for PDE4 enzymes, may increase in rolipramtreated cells to such an extent that unwanted cAMP-dependent processesin those cells may also become activated. Examples of cAMP-dependenteffector proteins include the PKA family of kinases, some ion channelsand certain guanine nucleotide exchange factors such as cAMP-GEF1 and 2.This example demonstrates that treatment with rolipram does not byitself increase cellular cAMP levels in CHO cells expressingHSPDE4A4B-GFP, although this fusion protein has phophodiesteraseactivity. Unwanted activation of cAMP-dependent processes in these cellsis therefore highly unlikely when rolipram is used to stimulate theproduction of the dense aggregates of PDE4A4 in these cells. CHO cellsstably expressing HSPDE4A4B-GFP (see Example 2), and untransfected CHOcells are seeded into individual wells of a 96-well microtiter plate ata density of 1.2×10⁵ cells/well, and cultured for 16 to 18 hours in HAMF-12 medium with glutamax, 100 μg penicillin-streptomycin mixture ml⁻¹and 10% FBS plus various concentrations of rolipram. After theincubation period, cAMP content is measured using a commercial kit fromAmersham-Pharmacia Biotech, kit # RPA538, according to the manufacturersinstructions. The amount of cAMP/well was calculated using regressionanalysis from a cAMP standrad curve, as recommended by the manufacturersof the assay kit. The results of this experiment are shown in FIG. 19.The curves for both normal untransfected CHO cells (♦), and cells stablyexpressing HSPDE4A4B-GFP (□) show no appreciable increase in cellularcAMP concentration over a range of rolipram treatments from 0.03 μM to 3μM. The slight rise in cAMP in normal CHO cells at 10 μM rolipram isprobably not significant, since this concentration is between 30 to 300fold higher than any reported IC₅₀ value for inhibition of any knownisoform of PDE4 by rolipram. In a separate experiment, untransfected CHOcells and CHO cells stably expressing HSPDE4A4B-GFP were cultured asdescribed for FIG. 19 without the addition of rolipram, but with variousconcentrations of forskolin added 60 minutes prior to measurement ofcAMP levels. The results of this experiment are shown in FIG. 20. Thecurve for normal untransfected CHO cells (♦) shows the expecteddose-dependent increase of cellular cAMP in response to this adenylatecyclase activator. Cells stably expressing HSPDE4A4B-GFP (□) show noappreciable increase in cellular cAMP concentration over a range offorskolin treatments from 0.3 μM to 100 μM. This result indicates thatthe HSPDE4A4-GFP fusion protein is an active phosphodiesterase enzymeand that its greatly increased expression effectively prevents any cAMPincrease in these cells despite adenylate cyclase activation byforskolin over the range tested.

[0260] Figure Legends

[0261]FIG. 1

[0262] Confocal fluorescence image showing CHO cells stably transfectedwith probe HSPDE4A4-EGFP-N1 growing in HAM's F12 medium with 10% FBS.The transfected cells are clonal, derived from a mixed, non-clonalpopulation. GFP fluorescence is more or less evenly distributedthroughout the non-nuclear cytoplasm, darker regions within this areaare probably mitochondria from which the probe is apparently excluded.

[0263]FIG. 2

[0264] Confocal fluorescence image showing CHO cells stably transfectedwith probe HSPDE4A4-EGFP-N1 growing in HAM's F12 medium with 10% FBS andwith 2 μM rolipram. The transfected cells have been derived from asingle cell isolated from a non-clonal population. The cells have beentreated with rolipram for 6.7 hours. GFP fluorescence concentrates inbright spots in more than 95% of the cells.

[0265]FIG. 3

[0266] Dose response of RP73401 versus 3 μM rolipram in clonal cellsexpressing HSPDE4A4-EGFP. Cells have been seeded into a 96-well PackardViewPlate and grown in HAM's F12 medium plus 10% FBS plus 3 μM rolipramto approximately 80% confluence. rolipram treatment was for a total of16 hours. Various concentrations of RP73401 were added to therolipram-containing wells and incubated for a further 4 hours. The cellwere then chemically fixed, washed and stained with Hoechst 33258, and ameasurement of mean spots per cell made for each treatment well using anautomated imager, all as described in Example 2. Each point is theaverage of the mean spots per cell determined from 5 individualtreatment wells at the same RP73401 concentration. Results have beenfitted to a 4-parameter Hill plot, and yield an IC₅₀ figure of 20 nM forthe competitive inhibition by RP73401 of spot formation by 3 μMrolipram.

[0267]FIG. 4

[0268] Dose response of RP73401 versus 1 μM rolipram in clonal cellsexpressing HSPDE4A4-EGFP, measured on the FLIPR. Cells have been seededinto a 96-well Packard ViewPlate and grown in HAM's F12 medium plus 10%FBS plus 1 μM rolipram to approximately 80% confluence. rolipramtreatment was for a total of 16 hours. Various concentrations of RP73401were added to the rolipram-containing wells and incubated for a further4 hours. The cells were then simultaneously chemically fixed andpermeabilised, washed and a measure of spot formation made on a FLIPRdevice, all as described in Example 2. The degree of spot formation(ordinate values) is given as raw values, which include the backgroundsignal (value from cells with no rolipram treatment)±standard deviation.Results have been fitted to a 4-parameter Hill plot, and yield an IC₅₀figure of approximately 50 nM for the competitive inhibition by RP73401of spot formation by 1 μM rolipram.

[0269]FIG. 5

[0270] Time-lapse images of CHO cells expressing HSPKAcat-EGFP, thefusion of EGFP to the C-terminal of human cAMP-dependent protein kinasecatalytic subunit isoform alpha, treated with forskolin. Cells are aclonal cell line growing in HAM's F12 medium plus 10% FBS to which hasbeen added I micromolar forskolin at time t=0. Successive images showhow the fluorescently tagged catalytic subunit of PKA redistributes fromthe bright aggregates into the cytoplasm over a period of minutes ascAMP levels increase in the cells as a result of forskolin treatment.Scale bar=10 microns.

[0271]FIG. 6

[0272] Time-lapse images of CHO cells expressing Cys1-EGFP, the fusionof EGFP to the C-terminal of the cys1 domain from human protein kinase Cisoform gamma, before (a) and after 5 minutes treatment with 100 nM PMA.Cells are a clonal cell line growing in HAM's F12 medium plus 10% FBS.After 5 minutes treatment with 100 nM PMA (b), the fluorescently taggedCys1 domain of PKCgamma redistributes from the cytoplasm and nucleus tothe plasma membrane.

[0273]FIG. 7

[0274] A clonal CHO cell line stably expressing the Cys1 domain of thehuman isoform of PKC gamma tagged with GFP in its C-terminal end wascultured in 96-well microliter plates (Packard View-Plate). After 20 minpreincubation in KRW (experimental buffer; A modified Krebs-Ringerbuffer containing (in mM): NaCl 140, KCl 3.6, NaH2PO4 0.5, MgSO4 0.5,NaHCO3 2.0, CaCl2 1.5 and HEPES 10, D-glucose 5 (pH 7.4, btrated with 1MNaOH)) the cell plate was placed in the FLIPR. Subsequently the cellswere stimulated in place on the FLIPR machine with the phorbol ester PMAgiven in doses from 10 μM to 0.5 nM in 1:3 steps. This gives a type ofredistribution response in these cells that can be quantified as areduction in fluorescence intensity, as PKCgamma-Cys1 moves from thecytosolic and nuclear compartments to the plasma membrane during thefirst 1-5 minutes of stimulation. The figure shows the dose response asmean (+SD) accumulated fluorescence change during the first threeminutes of stimulation with PMA (n=8). All data were corrected forbackground by subtraction of a background trace based on 8 individualbackground traces and set to zero at the point of addition of PMA.

[0275]FIG. 8

[0276] Confocal fluorescence images showing CHO cells stablyco-transfected with anchor probe HSPDE4A4-SosA and detectable probehGrb2-EGFP growing in HAM's F12 medium with 10% FBS plus (a) and similarcells after treatment with 10 μM rolipram (b). The transfected cells area mixed non-clonal population. The GFP-labelled detectable probe (GFPfluorescence) is distributed throughout the cell prior to rolipramtreatment (a), with a slightly higher concentration in the nuclei. Afterrolipram treatment (20 hours; b) some cells, in clonal groups, developdistinct bright spots. The appearance of the spots, and the requirementfor rolipram, indicate that in these cells the anchor probe hasresponded to rolipram as PDE4A4 is expected to (FIG. 2), and that theGFP-labelled detectable probe must be associated with it.

[0277]FIG. 9

[0278] The same population of cells as in FIG. 8b, treated for 20 hourswith 10 micromolar rolipram (a) are additionally treated with 10 μMRP73401 for 75 minutes at room temperature (b). This treatment dissolvesrolipram-induced spots of HSPDE4A4-EGFP (FIGS. 3 and 4), and has thesame effect on GFP-bright spots in these cells, confirming that thesespots are the result of interaction between the anchor probe andGFP-labelled detectable probe.

[0279]FIG. 10

[0280] CHO cells expressing HSPDE4A4-EGFP have been treated with 10 μMrolipram for 20 hours then chemically fixed and immunostained with anantibody raised in an ovine host against a peptide identical to aportion of the unique C-terminal sequence of HSPDE4A enzymes. Theantibody is detected using Alexa 549-conjugated donkey anti-sheepantibody (Molecular Probes Inc., Oregon, USA). This pair of confocalmicrographs show the image of EGFP fluorescence in the cells (a) incomparison to that obtained for Alexa 549 fluorescence (b). Emissionfilters (a: 515-525 nm bandpass. b: 590 nm long pass) have been chosento ensure that there is no significant bleed-through of fluorescencebetween GFP and Alexa 594 images. These images demonstrate how thePDE4A4 antibody can be used to detect aggregates of HSPDE4A4independently of the need for an EGFP tag, which is useful in confirmingthe existence of unlabelled HSPDE4A4-based anchor probes inco-transfected cells that have been rolipram treated.

[0281]FIG. 11

[0282] CHO cells stably expressing both the anchor probe HSPKAcat-hGrb2and GFP-labelled detectable probe EGFP-Sos-Cterm before (a) and 10minutes after (b) treatment with 50 micromolar forskolin+500 micromolarIBMX. Three cells are evident in (a) with GFP-bright aggregates typicalof the distribution of PKAcat-EGFP seen in unstimulated cells (FIG. 5,t=0 minutes). The dispersal of these spots by forskolin+IBMX, atreatment which increases cAMP in the cells, shows that the GFP-labelleddetectable probe must be interacting with the anchor probe in theco-transfected cells, because only the anchor probe can react toincreased cAMP in this way (FIG. 5).

[0283]FIG. 12

[0284] CHO cells stably expressing both the anchor probe Cys1-hGrb2 andGFP-labelled detectable probe EGFP-Sos-Cterm before (a) and 5 minutesafter (b) treatment with 100 nM PMA. The majority of cells in (a) have arather general cytoplasmic and nuclear distribution of fluorescence.Treatment with PMA causes a measurable shift in fluorescence to theplasma membrane, in the same way that Cys1-EGFP would redistribute inresponse to PMA (FIG. 6). This result shows that the GFP-labelleddetectable probe must be interacting with the anchor probe, because onlythe anchor probe can react to PMA in this way.

[0285]FIG. 13

[0286] Confocal fluorescence images showing CHO cells stably transfectedwith probe HSPDE4A1-EGFP. Images are recorded at the same microscopesettings for direct comparison of intensities. The transfected cells area clonal population derived from a single parent cell. In FIG. 13a thecells are growing in only HAM's F12 medium with 10% FBS; the GFPfluorescence is restricted to bright granule-like spots within theperinuclear cytoplasm of each cell. In FIG. 13b similar cells to thoseseen in FIG. 13a have been treated with 2 micromolar rolipram for 2hours. The majority of GFP-bright spots disappear in all cells underrolipram treatment, and the cytoplasm becomes generally brighter. Largerspots do not disperse in some cells. When rolipram is washed away, thespots reform within 1.75 hours.

[0287]FIG. 14

[0288]FIG. 14 shows a dose response curve for PDE4A1 spot dispersal inresponse to rolipram. The number of spots per cell for eachconcentration of the different inhibitors is the mean of 4measurements±sem, where each measurement is itself an average taken fromnot less than 100 cells. Cells are grown in HAM's F12 medium plus 10%FBS plus various concentrations of rolipram for 7 hours. The cells arethen chemically fixed with 4% formalin buffer (pH7) for 15 minutes,washed with PBS and stained with 10 μM Hoechst 33258 in PBS for 10minutes at 25° C., then washed twice in PBS. Automated images arecollected and analysed for the number of spots per cell as described inExample 2.

[0289]FIG. 15

[0290] Effect of stimulating CHO cells expressing an in frame proteinfusion between PDE4A4 and human 14-3-3beta and an in frame proteinfusion between EGFP and human BAD. A marked appearance of spots isobserved in the cells stimulated with rolipram (left panels). Extracttedcells retain most of the fluorescence that is located in spots andaggregates, but lose that which is more generally distributed in thecytoplasm. This non-anchored fluorescence is presumably more mobile, andtherefore rapidly washes out of the cells upon their permeabilisation.

[0291]FIG. 16

[0292] Effect of stimulating CHO cells expressing the NFkB(p65)EGFPfusion with ll -1 at 1 ng/ml. A marked redistribution from the cytosolto the nucleus is observed (left panel). Extracted cells retain most ofthe fluorescence originating from the nucleus, whereas fluorescenceoriginating from the cytosol is lost (right panel).

[0293]FIG. 17

[0294] Trap assay (spots): 4A4-14-3-3b+eGFP-BAD

[0295] A: the absolute fluorescence measured.

[0296] B: the signal to noise ratio in based on the data in figure A. Ascan be seen, the optimal signal to noise ratio is in this case obtainedwith Formalin 0.8%+Triton-x100 2%.

[0297]FIG. 18

[0298] Dose response on the effect of forskolin to stimulate PKAtranslocation. The calculated EC₅₀=3.28uM forskolin.

[0299]FIG. 19

[0300] CHO cells stably expressing HSPDE4A4B-GFP (see Example 2), anduntransfected CHO cells are seeded into individual wells of a 96-wellmicrotiter plate at a density of 1.2×10⁵ cells/well, and cultured for 16to 18 hours in HAM F-12 medium with glutamax, 100 μgpenicillin-streptomycin mixture ml⁻¹ and 10% FBS plus variousconcentrations of rolipram. After the incubation period, cAMP content ismeasured using a commercial kit from Amersham-Pharnacia Biotech, kit #RPA538, according to the manufacturers instructions. The amount ofcAMP/well was calculated using regression analysis from a CAMP standradcurve, as recommended by the manufacturers of the assay kit. The curvesfor both normal untransfected CHO cells (♦), and cells stably expressingHSPDE4A4B-GFP (□) show no appreciable increase in cellular cAMPconcentration over a range of rolipram treatments from 0.03 μM to 3 μM.The slight rise in cAMP in normal CHO cells at 10 μM rolipram isprobably not significant.

[0301]FIG. 20

[0302] CHO cells stably expressing HSPDE4A4B-GFP (see Example 2), anduntransfected CHO cells are seeded into individual wells of a 96-wellmicrotiter plate at a density of 1.2×10⁵ cells/well, and cultured for 16to 18 hours in HAM F-12 medium with glutamax, 100 μgpenicillin-streptomycin mixture ml⁻¹ and 10% FBS. After 16 to 18 hoursincubation, cells are treated for 60 minutes with various concentrationsof forskolin. After the treatment period, cAMP content is measured usinga commercial kit from Amersham-Pharmacia Biotech, kit # RPA538,according to the manufacturers instructions. The amount of cAMP/well wascalculated using regression analysis from a cAMP standrad curve, asrecommended by the manufacturers of the assay kit. The curve for normaluntransfected CHO cells (♦*) shows the expected dose-dependent increaseof cellular cAMP in response to this adenylate cyclase activator. Cellsstably expressing HSPDE4A4B-GFP (□) show no appreciable increase incellular CAMP concentration over a range of forskolin treatments from0.3 μM to 100 μM.

1 29 1 2666 DNA Aequoria Victoria and Homo sapiens 1 atggaacccccgaccgtccc ctcggaaagg agcctgtctc tgtcactgcc cgggccccgg 60 gagggccaggccaccctgaa gcctcccccg cagcacctgt ggcggcagcc tcggaccccc 120 atccgtatccagcagcgcgg ctactccgac agcgcggagc gcgccgagcg ggagcggcag 180 ccgcaccggcccatagagcg cgccgatgcc atggacacca gcgaccggcc cggcctgcgc 240 acgacccgcatgtcctggcc ctcgtccttc catggcactg gcaccggcag cggcggcgcg 300 ggcggaggcagcagcaggcg cttcgaggca gagaatgggc cgacaccatc tcctggccgc 360 agccccctggactcgcaggc gagcccagga ctcgtgctgc acgccggggc ggccaccagc 420 cagcgccgggagtccttcct gtaccgctca gacagcgact atgacatgtc acccaagacc 480 atgtcccggaactcatcggt caccagcgag gcgcacgctg aagacctcat cgtaacacca 540 tttgctcaggtgctggccag cctccggagc gtccgtagca acttctcact cctgaccaat 600 gtgcccgttcccagtaacaa gcggtccccg ctgggcggcc ccacccctgt ctgcaaggcc 660 acgctgtcagaagaaacgtg tcagcagttg gcccgggaga ctctggagga gctggactgg 720 tgtctggagcagctggagac catgcagacc tatcgctctg tcagcgagat ggcctcgcac 780 aagtttaaaaggatgttgaa ccgtgagctc acacacctgt cagaaatgag caggtccgga 840 aaccaggtctcagagtacat ttccacaaca ttcctggaca aacagaatga agtggagatc 900 ccatcacccacgatgaagga acgagaaaaa cagcaagcgc cgcgaccaag accctcccag 960 ccgcccccgccccctgtacc acacttacag cccatgtccc aaatcacagg gttgaaaaag 1020 ttgatgcatagtaacagcct gaacaactct aacattcccc gatttggggt gaagaccgat 1080 caagaagagctcctggccca agaactggag aacctgaaca agtggggcct gaacatcttt 1140 tgcgtgtcggattacgctgg aggccgctca ctcacctgca tcatgtacat gatattccag 1200 gagcgggacctgctgaagaa attccgcatc ccggtggaca cgatggtgac atacatgctg 1260 acgctggaggatcactacca cgctgacgtg gcctaccata acagcctgca cgcagctgac 1320 gtgctgcagtccacccacgt actgctggcc acgcctgcac tagatgcagt gttcacggac 1380 ctggagattctcgccgccct cttcgcggct gccatccacg atgtggatca ccctggggtc 1440 tccaaccagttcctcatcaa caccaattcg gagctggcgc tcatgtacaa cgatgagtcg 1500 gtgctcgagaatcaccacct ggccgtgggc ttcaagctgc tgcaggagga caactgcgac 1560 atcttccagaacctcagcaa gcgccagcgg cagagcctac gcaagatggt catcgacatg 1620 gtgctggccacggacatgtc caagcacatg accctcctgg ctgacctgaa gaccatggtg 1680 gagaccaagaaagtgaccag ctcaggggtc ctcctgctag ataactactc cgaccgcatc 1740 caggtcctccggaacatggt gcactgtgcc gacctcagca accccaccaa gccgctggag 1800 ctgtaccgccagtggacaga ccgcatcatg gccgagttct tccagcaggg tgaccgagag 1860 cgcgagcgtggcatggaaat cagccccatg tgtgacaagc acactgcctc cgtggagaag 1920 tctcaggtgggttttattga ctacattgtg cacccattgt gggagacctg ggcggacctt 1980 gtccacccagatgcccagga gatcttggac actttggagg acaaccggga ctggtactac 2040 agcgccatccggcagagccc atctccgcca cccgaggagg agtcaagggg gccaggccac 2100 ccacccctgcctgacaagtt ccagtttgag ctgacgctgg aggaggaaga ggaggaagaa 2160 atatcaatggcccagatacc gtgcacagcc caagaggcat tgactgcgca gggattgtca 2220 ggagtcgaggaagctctgga tgcaaccata gcctgggagg catccccggc ccaggagtcg 2280 ttggaagttatggcacagga agcatccctg gaggccgagc tggaggcagt gtatttgaca 2340 cagcaggcacagtccacagg cagtgcacct gtggctccgg atgagttctc gtcccgggag 2400 gaattcgtggttgctgtaag ccacagcagc ccctctgccc tggctcttca aagccccctt 2460 ctccctgcttggaggaccct gtctgtttca gagcatgccc cgggcctccc gggcctcccc 2520 tccacggcggccgaggtgga ggcccaacga gagcaccagg ctgccaagag ggcttgcagt 2580 gcctgcgcagggacatttgg ggaggacaca tccgcactcc cagctcctgg tggcgggggg 2640 tcaggtggagaccctacctg ggatcc 2666 2 886 PRT Aequoria victoria and Homo sapiens 2Met Glu Pro Pro Thr Val Pro Ser Glu Arg Ser Leu Ser Leu Ser Leu 1 5 1015 Pro Gly Pro Arg Glu Gly Gln Ala Thr Leu Lys Pro Pro Pro Gln His 20 2530 Leu Trp Arg Gln Pro Arg Thr Pro Ile Arg Ile Gln Gln Arg Gly Tyr 35 4045 Ser Asp Ser Ala Glu Arg Ala Glu Arg Glu Arg Gln Pro His Arg Pro 50 5560 Ile Glu Arg Ala Asp Ala Met Asp Thr Ser Asp Arg Pro Gly Leu Arg 65 7075 80 Thr Thr Arg Met Ser Trp Pro Ser Ser Phe His Gly Thr Gly Thr Gly 8590 95 Ser Gly Gly Ala Gly Gly Gly Ser Ser Arg Arg Phe Glu Ala Glu Asn100 105 110 Gly Pro Thr Pro Ser Pro Gly Arg Ser Pro Leu Asp Ser Gln AlaSer 115 120 125 Pro Gly Leu Val Leu His Ala Gly Ala Ala Thr Ser Gln ArgArg Glu 130 135 140 Ser Phe Leu Tyr Arg Ser Asp Ser Asp Tyr Asp Met SerPro Lys Thr 145 150 155 160 Met Ser Arg Asn Ser Ser Val Thr Ser Glu AlaHis Ala Glu Asp Leu 165 170 175 Ile Val Thr Pro Phe Ala Gln Val Leu AlaSer Leu Arg Ser Val Arg 180 185 190 Ser Asn Phe Ser Leu Leu Thr Asn ValPro Val Pro Ser Asn Lys Arg 195 200 205 Ser Pro Leu Gly Gly Pro Thr ProVal Cys Lys Ala Thr Leu Ser Glu 210 215 220 Glu Thr Cys Gln Gln Leu AlaArg Glu Thr Leu Glu Glu Leu Asp Trp 225 230 235 240 Cys Leu Glu Gln LeuGlu Thr Met Gln Thr Tyr Arg Ser Val Ser Glu 245 250 255 Met Ala Ser HisLys Phe Lys Arg Met Leu Asn Arg Glu Leu Thr His 260 265 270 Leu Ser GluMet Ser Arg Ser Gly Asn Gln Val Ser Glu Tyr Ile Ser 275 280 285 Thr ThrPhe Leu Asp Lys Gln Asn Glu Val Glu Ile Pro Ser Pro Thr 290 295 300 MetLys Glu Arg Glu Lys Gln Gln Ala Pro Arg Pro Arg Pro Ser Gln 305 310 315320 Pro Pro Pro Pro Pro Val Pro His Leu Gln Pro Met Ser Gln Ile Thr 325330 335 Gly Leu Lys Lys Leu Met His Ser Asn Ser Leu Asn Asn Ser Asn Ile340 345 350 Pro Arg Phe Gly Val Lys Thr Asp Gln Glu Glu Leu Leu Ala GlnGlu 355 360 365 Leu Glu Asn Leu Asn Lys Trp Gly Leu Asn Ile Phe Cys ValSer Asp 370 375 380 Tyr Ala Gly Gly Arg Ser Leu Thr Cys Ile Met Tyr MetIle Phe Gln 385 390 395 400 Glu Arg Asp Leu Leu Lys Lys Phe Arg Ile ProVal Asp Thr Met Val 405 410 415 Thr Tyr Met Leu Thr Leu Glu Asp His TyrHis Ala Asp Val Ala Tyr 420 425 430 His Asn Ser Leu His Ala Ala Asp ValLeu Gln Ser Thr His Val Leu 435 440 445 Leu Ala Thr Pro Ala Leu Asp AlaVal Phe Thr Asp Leu Glu Ile Leu 450 455 460 Ala Ala Leu Phe Ala Ala AlaIle His Asp Val Asp His Pro Gly Val 465 470 475 480 Ser Asn Gln Phe LeuIle Asn Thr Asn Ser Glu Leu Ala Leu Met Tyr 485 490 495 Asn Asp Glu SerVal Leu Glu Asn His His Leu Ala Val Gly Phe Lys 500 505 510 Leu Leu GlnGlu Asp Asn Cys Asp Ile Phe Gln Asn Leu Ser Lys Arg 515 520 525 Gln ArgGln Ser Leu Arg Lys Met Val Ile Asp Met Val Leu Ala Thr 530 535 540 AspMet Ser Lys His Met Thr Leu Leu Ala Asp Leu Lys Thr Met Val 545 550 555560 Glu Thr Lys Lys Val Thr Ser Ser Gly Val Leu Leu Leu Asp Asn Tyr 565570 575 Ser Asp Arg Ile Gln Val Leu Arg Asn Met Val His Cys Ala Asp Leu580 585 590 Ser Asn Pro Thr Lys Pro Leu Glu Leu Tyr Arg Gln Trp Thr AspArg 595 600 605 Ile Met Ala Glu Phe Phe Gln Gln Gly Asp Arg Glu Arg GluArg Gly 610 615 620 Met Glu Ile Ser Pro Met Cys Asp Lys His Thr Ala SerVal Glu Lys 625 630 635 640 Ser Gln Val Gly Phe Ile Asp Tyr Ile Val HisPro Leu Trp Glu Thr 645 650 655 Trp Ala Asp Leu Val His Pro Asp Ala GlnGlu Ile Leu Asp Thr Leu 660 665 670 Glu Asp Asn Arg Asp Trp Tyr Tyr SerAla Ile Arg Gln Ser Pro Ser 675 680 685 Pro Pro Pro Glu Glu Glu Ser ArgGly Pro Gly His Pro Pro Leu Pro 690 695 700 Asp Lys Phe Gln Phe Glu LeuThr Leu Glu Glu Glu Glu Glu Glu Glu 705 710 715 720 Ile Ser Met Ala GlnIle Pro Cys Thr Ala Gln Glu Ala Leu Thr Ala 725 730 735 Gln Gly Leu SerGly Val Glu Glu Ala Leu Asp Ala Thr Ile Ala Trp 740 745 750 Glu Ala SerPro Ala Gln Glu Ser Leu Glu Val Met Ala Gln Glu Ala 755 760 765 Ser LeuGlu Ala Glu Leu Glu Ala Val Tyr Leu Thr Gln Gln Ala Gln 770 775 780 SerThr Gly Ser Ala Pro Val Ala Pro Asp Glu Phe Ser Ser Arg Glu 785 790 795800 Glu Phe Val Val Ala Val Ser His Ser Ser Pro Ser Ala Leu Ala Leu 805810 815 Gln Ser Pro Leu Leu Pro Ala Trp Arg Thr Leu Ser Val Ser Glu His820 825 830 Ala Pro Gly Leu Pro Gly Leu Pro Ser Thr Ala Ala Glu Val GluAla 835 840 845 Gln Arg Glu His Gln Ala Ala Lys Arg Ala Cys Ser Ala CysAla Gly 850 855 860 Thr Phe Gly Glu Asp Thr Ser Ala Leu Pro Ala Pro GlyGly Gly Gly 865 870 875 880 Ser Gly Gly Asp Pro Thr 885 3 1949 DNAAequoria victoria and Homo sapiens 3 atgcccttgg tggatttctt ctgcgagacctgctctaagc cttggctggt gggctggtgg 60 gaccagttta aaaggatgtt gaaccgtgagctcacacacc tgtcagaaat gagcaggtcc 120 ggaaaccagg tctcagagta catttccacaacattcctgg acaaacagaa tgaagtggag 180 atcccatcac ccacgatgaa ggaacgagaaaaacagcaag cgccgcgacc aagaccctcc 240 cagccgcccc cgccccctgt accacacttacagcccatgt cccaaatcac agggttgaaa 300 aagttgatgc atagtaacag cctgaacaactctaacattc cccgatttgg ggtgaagacc 360 gatcaagaag agctcctggc ccaagaactggagaacctga acaagtgggg cctgaacatc 420 ttttgcgtgt cggattacgc tggaggccgctcactcacct gcatcatgta catgatattc 480 caggagcggg acctgctgaa gaaattccgcatcccggtgg acacgatggt gacatacatg 540 ctgacgctgg aggatcacta ccacgctgacgtggcctacc ataacagcct gcacgcagct 600 gacgtgctgc agtccaccca cgtactgctggccacgcctg cactagatgc agtgttcacg 660 gacctggaga ttctcgccgc cctcttcgcggctgccatcc acgatgtgga tcaccctggg 720 gtctccaacc agttcctcat caacaccaattcggagctgg cgctcatgta caacgatgag 780 tcggtgctcg agaatcacca cctggccgtgggcttcaagc tgctgcagga ggacaactgc 840 gacatcttcc agaacctcag caagcgccagcggcagagcc tacgcaagat ggtcatcgac 900 atggtgctgg ccacggacat gtccaagcacatgaccctcc tggctgacct gaagaccatg 960 gtggagacca agaaagtgac cagctcaggggtcctcctgc tagataacta ctccgaccgc 1020 atccaggtcc tccggaacat ggtgcactgtgccgacctca gcaaccccac caagccgctg 1080 gagctgtacc gccagtggac agaccgcatcatggccgagt tcttccagca gggtgaccga 1140 gagcgcgagc gtggcatgga aatcagccccatgtgtgaca agcacactgc ctccgtggag 1200 aagtctcagg tgggttttat tgactacattgtgcacccat tgtgggagac ctgggcggac 1260 cttgtccacc cagatgccca ggagatcttggacactttgg aggacaaccg ggactggtac 1320 tacagcgcca tccggcagag cccatctccgccacccgagg aggagtcaag ggggccaggc 1380 cacccacccc tgcctgacaa gttccagtttgagctgacgc tggaggagga agaggaggaa 1440 gaaatatcaa tggcccagat accgtgcacagcccaagagg cattgactgc gcagggattg 1500 tcaggagtcg aggaagctct ggatgcaaccatagcctggg aggcatcccc ggcccaggag 1560 tcgttggaag ttatggcaca ggaagcatccctggaggccg agctggaggc agtgtatttg 1620 acacagcagg cacagtccac aggcagtgcacctgtggctc cggatgagtt ctcgtcccgg 1680 gaggaattcg tggttgctgt aagccacagcagcccctctg ccctggctct tcaaagcccc 1740 cttctccctg cttggaggac cctgtctgtttcagagcatg ccccgggcct cccgggcctc 1800 ccctccacgg cggccgaggt ggaggcccaacgagagcacc aggctgccaa gagggcttgc 1860 agtgcctgcg cagggacatt tggggaggacacatccgcac tcccagctcc tggtggcggg 1920 gggtcaggtg gagaccctac ctgggatcc1949 4 647 PRT Aequoria victoria and Homo sapiens 4 Met Pro Leu Val AspPhe Phe Cys Glu Thr Cys Ser Lys Pro Trp Leu 1 5 10 15 Val Gly Trp TrpAsp Gln Phe Lys Arg Met Leu Asn Arg Glu Leu Thr 20 25 30 His Leu Ser GluMet Ser Arg Ser Gly Asn Gln Val Ser Glu Tyr Ile 35 40 45 Ser Thr Thr PheLeu Asp Lys Gln Asn Glu Val Glu Ile Pro Ser Pro 50 55 60 Thr Met Lys GluArg Glu Lys Gln Gln Ala Pro Arg Pro Arg Pro Ser 65 70 75 80 Gln Pro ProPro Pro Pro Val Pro His Leu Gln Pro Met Ser Gln Ile 85 90 95 Thr Gly LeuLys Lys Leu Met His Ser Asn Ser Leu Asn Asn Ser Asn 100 105 110 Ile ProArg Phe Gly Val Lys Thr Asp Gln Glu Glu Leu Leu Ala Gln 115 120 125 GluLeu Glu Asn Leu Asn Lys Trp Gly Leu Asn Ile Phe Cys Val Ser 130 135 140Asp Tyr Ala Gly Gly Arg Ser Leu Thr Cys Ile Met Tyr Met Ile Phe 145 150155 160 Gln Glu Arg Asp Leu Leu Lys Lys Phe Arg Ile Pro Val Asp Thr Met165 170 175 Val Thr Tyr Met Leu Thr Leu Glu Asp His Tyr His Ala Asp ValAla 180 185 190 Tyr His Asn Ser Leu His Ala Ala Asp Val Leu Gln Ser ThrHis Val 195 200 205 Leu Leu Ala Thr Pro Ala Leu Asp Ala Val Phe Thr AspLeu Glu Ile 210 215 220 Leu Ala Ala Leu Phe Ala Ala Ala Ile His Asp ValAsp His Pro Gly 225 230 235 240 Val Ser Asn Gln Phe Leu Ile Asn Thr AsnSer Glu Leu Ala Leu Met 245 250 255 Tyr Asn Asp Glu Ser Val Leu Glu AsnHis His Leu Ala Val Gly Phe 260 265 270 Lys Leu Leu Gln Glu Asp Asn CysAsp Ile Phe Gln Asn Leu Ser Lys 275 280 285 Arg Gln Arg Gln Ser Leu ArgLys Met Val Ile Asp Met Val Leu Ala 290 295 300 Thr Asp Met Ser Lys HisMet Thr Leu Leu Ala Asp Leu Lys Thr Met 305 310 315 320 Val Glu Thr LysLys Val Thr Ser Ser Gly Val Leu Leu Leu Asp Asn 325 330 335 Tyr Ser AspArg Ile Gln Val Leu Arg Asn Met Val His Cys Ala Asp 340 345 350 Leu SerAsn Pro Thr Lys Pro Leu Glu Leu Tyr Arg Gln Trp Thr Asp 355 360 365 ArgIle Met Ala Glu Phe Phe Gln Gln Gly Asp Arg Glu Arg Glu Arg 370 375 380Gly Met Glu Ile Ser Pro Met Cys Asp Lys His Thr Ala Ser Val Glu 385 390395 400 Lys Ser Gln Val Gly Phe Ile Asp Tyr Ile Val His Pro Leu Trp Glu405 410 415 Thr Trp Ala Asp Leu Val His Pro Asp Ala Gln Glu Ile Leu AspThr 420 425 430 Leu Glu Asp Asn Arg Asp Trp Tyr Tyr Ser Ala Ile Arg GlnSer Pro 435 440 445 Ser Pro Pro Pro Glu Glu Glu Ser Arg Gly Pro Gly HisPro Pro Leu 450 455 460 Pro Asp Lys Phe Gln Phe Glu Leu Thr Leu Glu GluGlu Glu Glu Glu 465 470 475 480 Glu Ile Ser Met Ala Gln Ile Pro Cys ThrAla Gln Glu Ala Leu Thr 485 490 495 Ala Gln Gly Leu Ser Gly Val Glu GluAla Leu Asp Ala Thr Ile Ala 500 505 510 Trp Glu Ala Ser Pro Ala Gln GluSer Leu Glu Val Met Ala Gln Glu 515 520 525 Ala Ser Leu Glu Ala Glu LeuGlu Ala Val Tyr Leu Thr Gln Gln Ala 530 535 540 Gln Ser Thr Gly Ser AlaPro Val Ala Pro Asp Glu Phe Ser Ser Arg 545 550 555 560 Glu Glu Phe ValVal Ala Val Ser His Ser Ser Pro Ser Ala Leu Ala 565 570 575 Leu Gln SerPro Leu Leu Pro Ala Trp Arg Thr Leu Ser Val Ser Glu 580 585 590 His AlaPro Gly Leu Pro Gly Leu Pro Ser Thr Ala Ala Glu Val Glu 595 600 605 AlaGln Arg Glu His Gln Ala Ala Lys Arg Ala Cys Ser Ala Cys Ala 610 615 620Gly Thr Phe Gly Glu Asp Thr Ser Ala Leu Pro Ala Pro Gly Gly Gly 625 630635 640 Gly Ser Gly Gly Asp Pro Thr 645 5 3999 DNA Aequoria Victoria andHomo sapiens 5 atgcagcagg cgccgcagcc ttacgagttc ttcagcgagg agaacagtccgaaatggcgg 60 ggactgttgg tctcggccct gcggaaggtt caggaacaag tgcatcccactctctcagct 120 aatgaagagt ctctctatta tattgaagag ctgatttttc cgctgcttaataaattatgc 180 gtggcccagc caagcactgt tcaagatgta gaggagcgag ttcagaagacctttcctcac 240 ccaattgata aatgggccat tgctgatgca caatctgcta tagaaaaacgaaaacgaaga 300 aatcctcttt tactgcctgt ggacaaaatc catccttcgt tgaaggaagtattagggtac 360 aaagtggact accatgtatc cctatatatt gtggctgtac tagagtatatctcagctgat 420 attttaaaat tggctggtaa ttatgttttt aatatccggc attatgaaatatctcagcag 480 gacattaaag tgtcaatgtg tgcggataag gttttgatgg acatgtttgatcaggatgac 540 ataggtttgg tttctctctg tgaagatgaa cctggttctt ctggtgaattaaactactat 600 gatcttgtca gaactgaaat cgcagaagaa agacagtatc tacgggaattaaatatgatc 660 ataaaagtgt ttcgagaagc ctttctttct gatagaaagc tgtttaaaccttctgatatc 720 gaaaagattt ttagtaacat ttcagatata catgaattga ctgtgaaacttttaggtttg 780 attgaagaca cagttgaaat gactgatgaa agcagtcctc atcccttagctggcagctgt 840 tttgaagatt tggcagaaga gcaagcattt gatccttatg aaacattatcacaggacatt 900 ctttcaccag agtttcatga acatttcaat aaattgatgg ccagacctgcagttgctcta 960 cactttcagt ccattgctga tggttttaaa gaggcagttc gttacgtccttccacgtctt 1020 atgctggtgc cagtgtatca ctgttggcac tactttgagt tactaaagcaattgaaagca 1080 tgtagtgaag aacaagaaga cagagaatgt ttgaaccaag ctattactgctctcatgaat 1140 ctccaaggta gcatggaccg aatttacaag cagtattcac ctagacgtcgacctggagat 1200 cctgtttgcc ctttttatag tcaccaatta agaagcaaac acctggctatcaaaaaaatg 1260 aatgaaattc agaaaaatat cgatggatgg gaaggcaaag atattggacagtgttgtaat 1320 gaattcatta tggagggacc attgacaaga atcggtgcca aacatgaacggcatattttt 1380 ctgtttgatg gcttaatgat cagttgtaaa cctaatcatg gccagactcggcttccaggt 1440 tacagtagtg cagaatacag gttaaaagaa aaatttgtca tgaggaaaatacaaatttgt 1500 gataaagaag atacttgtga gcacaagcat gcatttgaat tagtatccaaagatgagaac 1560 agcataatat ttgctgctaa gtctgctgaa gaaaaaaaca actggatggcagcccttatt 1620 tctcttcatt atcgtagtac tctagatcga atgttagatt cagtattattgaaagaagaa 1680 aatgagcaac cactgagatt accaagtcct gaagtatatc gttttgtagtaaaagactct 1740 gaggaaaaca ttgtttttga agacaacttg caaagtagaa gtggcatccccattattaaa 1800 ggaggaactg tagtgaaatt aattgaaagg ttaacatatc atatgtatgcagatcccaat 1860 tttgttcgta cttttcttac cacatatcgt tcattttgta aaccacaggaattgctgagc 1920 ttactgattg aacggtttga aattccagag ccagaaccta ctgacgcagacaaattggca 1980 atagagaaag gcgagcagcc aatcagtgca gaccttaaaa gatttcgcaaggaatatgtc 2040 caaccagtac aacttaggat cttaaatgta tttcggcatt gggttgaacatcatttttat 2100 gactttgaaa gagacttgga attgcttgaa agactagaat ccttcatttcaagtgtaaga 2160 gggaaagcta tgaaaaaatg ggtagagtca attgctaaga tcatcaggaggaagaagcaa 2220 gctcaggcaa atggagtaag ccataatatt acctttgaaa gtccacctccaccaattgaa 2280 tggcatatca gcaaaccagg acagtttgaa acatttgatc tcatgacacttgatccaata 2340 gaaattgcac gtcagctgac acttttggag tctgatcttt acaggaaagttcaaccgtct 2400 gaacttgtag ggagtgtgtg gaccaaagaa gataaagaaa taaattctccaaatttatta 2460 aaaatgattc gccataccac aaatctcacc ctctggtttg aaaaatgcattgtggaagca 2520 gaaaattttg aagaacgggt ggcagtacta agtagaatta tagaaattctgcaagttttt 2580 caagatttga ataatttcaa tggcgtattg gagatagtca gtgcagtaaattcagtgtca 2640 gtatacagac tagaccatac ctttgaggca ctgcaggaaa ggaaaaggaaaattttggac 2700 gaagctgtgg aattaagtca agatcacttt aaaaaatacc tagtaaaacttaagtcaatc 2760 aatccacctt gtgtgccttt ttttggaata tatttaacaa atattctgaagaccgaagaa 2820 gggaataatg attttttaaa aaagaaaggg aaagatttaa tcaatttcagtaagaggagg 2880 aaagtagctg aaattactgg agaaattcag cagtatcaga atcagccttactgtttacgg 2940 atagaaccag atatgaggag attctttgaa aaccttaacc ccatgggaagtgcatctgaa 3000 aaagagttta cagattattt gttcaacaag tcactagaaa ttgaacctcgaaactgcaaa 3060 cagccacctc gatttcctag gaaatcaact ttttccttaa aatctcctggaataaagcct 3120 aacacaggcc gacatggctc tacctcaggt actttacgag gtcacccaacaccattagaa 3180 agagaaccat gtaaaataag ctttagtcgg attgctgaaa ctgagctggaatcaacagtg 3240 tcagcaccaa cctctccaaa tacaccatct actccaccag tatctgcttcttcagacctt 3300 agtgtatctt tagatgtgga tctcaacagc tcctgtggca gcaatagcatctttgctcca 3360 gtgcttttgc cacattcaaa gtctttcttt agttcatgtg gtagtttacataaactaagt 3420 gaagagcccc tgattcctcc tcctcttcct cctcgaaaaa agtttgatcatgatgcttca 3480 aattccaagg gaaatatgaa atctgatgat gatcctcctg ctattccaccgagacagcct 3540 cctcctccaa aggtaaaacc cagagttcct gttcctactg gtgcatttgatgggcctctg 3600 catagtccac ctccgccacc accaagagat cctcttcctg atacccctccaccagttccc 3660 cttcggcctc cagaacactt tataaactgt ccatttaatc ttcagccacctccactgggg 3720 catcttcaca gagattcaga ctggctcaga gacattagta cgtgtccaaattcgccaagc 3780 actcctccta gcacaccctc tccaagggta ccgcgtcgat gctatgtgctcagttctagt 3840 cagaataatc ttgctcatcc tccagctccc cctgttccac caaggcagaattcaagccct 3900 catctgccaa aactgccacc aaagacttac aaacgggagc tttcgcaccccccattgtac 3960 agactgcctt tgctagaaaa tgcagaaact ccccaatga 3999 6 1333PRT Aequoria Victoria and Homo sapiens 6 Met Gln Gln Ala Pro Gln Pro TyrGlu Phe Phe Ser Glu Glu Asn Ser 1 5 10 15 Pro Lys Trp Arg Gly Leu LeuVal Ser Ala Leu Arg Lys Val Gln Glu 20 25 30 Gln Val His Pro Thr Leu SerAla Asn Glu Glu Ser Leu Tyr Tyr Ile 35 40 45 Glu Glu Leu Ile Phe Pro LeuLeu Asn Lys Leu Cys Val Ala Gln Pro 50 55 60 Ser Thr Val Gln Asp Val GluGlu Arg Val Gln Lys Thr Phe Pro His 65 70 75 80 Pro Ile Asp Lys Trp AlaIle Ala Asp Ala Gln Ser Ala Ile Glu Lys 85 90 95 Arg Lys Arg Arg Asn ProLeu Leu Leu Pro Val Asp Lys Ile His Pro 100 105 110 Ser Leu Lys Glu ValLeu Gly Tyr Lys Val Asp Tyr His Val Ser Leu 115 120 125 Tyr Ile Val AlaVal Leu Glu Tyr Ile Ser Ala Asp Ile Leu Lys Leu 130 135 140 Ala Gly AsnTyr Val Phe Asn Ile Arg His Tyr Glu Ile Ser Gln Gln 145 150 155 160 AspIle Lys Val Ser Met Cys Ala Asp Lys Val Leu Met Asp Met Phe 165 170 175Asp Gln Asp Asp Ile Gly Leu Val Ser Leu Cys Glu Asp Glu Pro Gly 180 185190 Ser Ser Gly Glu Leu Asn Tyr Tyr Asp Leu Val Arg Thr Glu Ile Ala 195200 205 Glu Glu Arg Gln Tyr Leu Arg Glu Leu Asn Met Ile Ile Lys Val Phe210 215 220 Arg Glu Ala Phe Leu Ser Asp Arg Lys Leu Phe Lys Pro Ser AspIle 225 230 235 240 Glu Lys Ile Phe Ser Asn Ile Ser Asp Ile His Glu LeuThr Val Lys 245 250 255 Leu Leu Gly Leu Ile Glu Asp Thr Val Glu Met ThrAsp Glu Ser Ser 260 265 270 Pro His Pro Leu Ala Gly Ser Cys Phe Glu AspLeu Ala Glu Glu Gln 275 280 285 Ala Phe Asp Pro Tyr Glu Thr Leu Ser GlnAsp Ile Leu Ser Pro Glu 290 295 300 Phe His Glu His Phe Asn Lys Leu MetAla Arg Pro Ala Val Ala Leu 305 310 315 320 His Phe Gln Ser Ile Ala AspGly Phe Lys Glu Ala Val Arg Tyr Val 325 330 335 Leu Pro Arg Leu Met LeuVal Pro Val Tyr His Cys Trp His Tyr Phe 340 345 350 Glu Leu Leu Lys GlnLeu Lys Ala Cys Ser Glu Glu Gln Glu Asp Arg 355 360 365 Glu Cys Leu AsnGln Ala Ile Thr Ala Leu Met Asn Leu Gln Gly Ser 370 375 380 Met Asp ArgIle Tyr Lys Gln Tyr Ser Pro Arg Arg Arg Pro Gly Asp 385 390 395 400 ProVal Cys Pro Phe Tyr Ser His Gln Leu Arg Ser Lys His Leu Ala 405 410 415Ile Lys Lys Met Asn Glu Ile Gln Lys Asn Ile Asp Gly Trp Glu Gly 420 425430 Lys Asp Ile Gly Gln Cys Cys Asn Glu Phe Ile Met Glu Gly Pro Leu 435440 445 Thr Arg Ile Gly Ala Lys His Glu Arg His Ile Phe Leu Phe Asp Gly450 455 460 Leu Met Ile Ser Cys Lys Pro Asn His Gly Gln Thr Arg Leu ProGly 465 470 475 480 Tyr Ser Ser Ala Glu Tyr Arg Leu Lys Glu Lys Phe ValMet Arg Lys 485 490 495 Ile Gln Ile Cys Asp Lys Glu Asp Thr Cys Glu HisLys His Ala Phe 500 505 510 Glu Leu Val Ser Lys Asp Glu Asn Ser Ile IlePhe Ala Ala Lys Ser 515 520 525 Ala Glu Glu Lys Asn Asn Trp Met Ala AlaLeu Ile Ser Leu His Tyr 530 535 540 Arg Ser Thr Leu Asp Arg Met Leu AspSer Val Leu Leu Lys Glu Glu 545 550 555 560 Asn Glu Gln Pro Leu Arg LeuPro Ser Pro Glu Val Tyr Arg Phe Val 565 570 575 Val Lys Asp Ser Glu GluAsn Ile Val Phe Glu Asp Asn Leu Gln Ser 580 585 590 Arg Ser Gly Ile ProIle Ile Lys Gly Gly Thr Val Val Lys Leu Ile 595 600 605 Glu Arg Leu ThrTyr His Met Tyr Ala Asp Pro Asn Phe Val Arg Thr 610 615 620 Phe Leu ThrThr Tyr Arg Ser Phe Cys Lys Pro Gln Glu Leu Leu Ser 625 630 635 640 LeuLeu Ile Glu Arg Phe Glu Ile Pro Glu Pro Glu Pro Thr Asp Ala 645 650 655Asp Lys Leu Ala Ile Glu Lys Gly Glu Gln Pro Ile Ser Ala Asp Leu 660 665670 Lys Arg Phe Arg Lys Glu Tyr Val Gln Pro Val Gln Leu Arg Ile Leu 675680 685 Asn Val Phe Arg His Trp Val Glu His His Phe Tyr Asp Phe Glu Arg690 695 700 Asp Leu Glu Leu Leu Glu Arg Leu Glu Ser Phe Ile Ser Ser ValArg 705 710 715 720 Gly Lys Ala Met Lys Lys Trp Val Glu Ser Ile Ala LysIle Ile Arg 725 730 735 Arg Lys Lys Gln Ala Gln Ala Asn Gly Val Ser HisAsn Ile Thr Phe 740 745 750 Glu Ser Pro Pro Pro Pro Ile Glu Trp His IleSer Lys Pro Gly Gln 755 760 765 Phe Glu Thr Phe Asp Leu Met Thr Leu AspPro Ile Glu Ile Ala Arg 770 775 780 Gln Leu Thr Leu Leu Glu Ser Asp LeuTyr Arg Lys Val Gln Pro Ser 785 790 795 800 Glu Leu Val Gly Ser Val TrpThr Lys Glu Asp Lys Glu Ile Asn Ser 805 810 815 Pro Asn Leu Leu Lys MetIle Arg His Thr Thr Asn Leu Thr Leu Trp 820 825 830 Phe Glu Lys Cys IleVal Glu Ala Glu Asn Phe Glu Glu Arg Val Ala 835 840 845 Val Leu Ser ArgIle Ile Glu Ile Leu Gln Val Phe Gln Asp Leu Asn 850 855 860 Asn Phe AsnGly Val Leu Glu Ile Val Ser Ala Val Asn Ser Val Ser 865 870 875 880 ValTyr Arg Leu Asp His Thr Phe Glu Ala Leu Gln Glu Arg Lys Arg 885 890 895Lys Ile Leu Asp Glu Ala Val Glu Leu Ser Gln Asp His Phe Lys Lys 900 905910 Tyr Leu Val Lys Leu Lys Ser Ile Asn Pro Pro Cys Val Pro Phe Phe 915920 925 Gly Ile Tyr Leu Thr Asn Ile Leu Lys Thr Glu Glu Gly Asn Asn Asp930 935 940 Phe Leu Lys Lys Lys Gly Lys Asp Leu Ile Asn Phe Ser Lys ArgArg 945 950 955 960 Lys Val Ala Glu Ile Thr Gly Glu Ile Gln Gln Tyr GlnAsn Gln Pro 965 970 975 Tyr Cys Leu Arg Ile Glu Pro Asp Met Arg Arg PhePhe Glu Asn Leu 980 985 990 Asn Pro Met Gly Ser Ala Ser Glu Lys Glu PheThr Asp Tyr Leu Phe 995 1000 1005 Asn Lys Ser Leu Glu Ile Glu Pro ArgAsn Cys Lys Gln Pro Pro Arg 1010 1015 1020 Phe Pro Arg Lys Ser Thr PheSer Leu Lys Ser Pro Gly Ile Lys Pro 1025 1030 1035 1040 Asn Thr Gly ArgHis Gly Ser Thr Ser Gly Thr Leu Arg Gly His Pro 1045 1050 1055 Thr ProLeu Glu Arg Glu Pro Cys Lys Ile Ser Phe Ser Arg Ile Ala 1060 1065 1070Glu Thr Glu Leu Glu Ser Thr Val Ser Ala Pro Thr Ser Pro Asn Thr 10751080 1085 Pro Ser Thr Pro Pro Val Ser Ala Ser Ser Asp Leu Ser Val SerLeu 1090 1095 1100 Asp Val Asp Leu Asn Ser Ser Cys Gly Ser Asn Ser IlePhe Ala Pro 1105 1110 1115 1120 Val Leu Leu Pro His Ser Lys Ser Phe PheSer Ser Cys Gly Ser Leu 1125 1130 1135 His Lys Leu Ser Glu Glu Pro LeuIle Pro Pro Pro Leu Pro Pro Arg 1140 1145 1150 Lys Lys Phe Asp His AspAla Ser Asn Ser Lys Gly Asn Met Lys Ser 1155 1160 1165 Asp Asp Asp ProPro Ala Ile Pro Pro Arg Gln Pro Pro Pro Pro Lys 1170 1175 1180 Val LysPro Arg Val Pro Val Pro Thr Gly Ala Phe Asp Gly Pro Leu 1185 1190 11951200 His Ser Pro Pro Pro Pro Pro Pro Arg Asp Pro Leu Pro Asp Thr Pro1205 1210 1215 Pro Pro Val Pro Leu Arg Pro Pro Glu His Phe Ile Asn CysPro Phe 1220 1225 1230 Asn Leu Gln Pro Pro Pro Leu Gly His Leu His ArgAsp Ser Asp Trp 1235 1240 1245 Leu Arg Asp Ile Ser Thr Cys Pro Asn SerPro Ser Thr Pro Pro Ser 1250 1255 1260 Thr Pro Ser Pro Arg Val Pro ArgArg Cys Tyr Val Leu Ser Ser Ser 1265 1270 1275 1280 Gln Asn Asn Leu AlaHis Pro Pro Ala Pro Pro Val Pro Pro Arg Gln 1285 1290 1295 Asn Ser SerPro His Leu Pro Lys Leu Pro Pro Lys Thr Tyr Lys Arg 1300 1305 1310 GluLeu Ser His Pro Pro Leu Tyr Arg Leu Pro Leu Leu Glu Asn Ala 1315 13201325 Glu Thr Pro Gln Glx 1330 7 28 DNA Artificial Sequence 4A-Ct-top PCRprimer used to amplify the C-terminal part HSPDE4A4 7 gtttaaaaggatgttgaacc gtgagctc 28 8 28 DNA Artificial Sequence 4A-bottom PCR primerused to amplify the C-terminal part HSPDE4A4 and also to amplify thecoding region of HSPDE4A1 8 gtggatccca ggtagggtct ccacctga 28 9 28 DNAArtificial Sequence 4A4-top PCR primer used to amplify the N-terminalpart of HSPDE4A4 9 gtaagcttgc gccatggaac ccccgacc 28 10 33 DNAArtificial Sequence 4A4N-bottom PCR primer used to amplify theN-terminal part of HSPDE4A4 10 ggttttaaac ttgtgcgagg ccatctcgct gac 3311 37 DNA Artificial Sequence 9658-top PCR primer contains the Avr2cloning sites and flanks the zeocin resistance gene including its E.coli promoter 11 tcctaggctg cagcacgtgt tgacaattaa tcatcgg 37 12 37 DNAArtificial Sequence 9655-bottom PCR primer-contains Avr2 cloning sitesand flanks the zeocin resistance gene including its E. coli promoter 12tcctaggtca gtcctgctcc tcggccacga agtgcac 37 13 34 DNA ArtificialSequence 0099-top PCR primer used to isolate the coding sequence ofhuman SOS1 from a human fetus or brain cDNA library 13 gttggatcccatgcagcagg cgccgcagcc ttac 34 14 35 DNA Artificial Sequence 0100-bottomPCR primer used to isolate the coding sequence of human SOS1 from ahuman fetus or brain cDNA library 14 gttgcggccg ctcattgggg agtttctgcattttc 35 15 27 DNA Artificial Sequence 0073-top PCR primer used toisolate the coding sequence of human GRB2 from a human fetus or brain orplacenta cDNA library 15 gcgaagcttt cagaatggaa gccatcg 27 16 26 DNAArtificial Sequence 0074-bottom PCR primer used to isolate the codingsequence of human GRB2 from a human fetus or brain or placenta cDNAlibrary 16 gccgaattcg gacgttccgg ttcacg 26 17 28 DNA Artificial Sequence9949 oligonucleotide used to ligate the vector fragment to createresulting plasmid PS587 17 ctagcattaa tacgactcac tataggga 28 18 28 DNAArtificial Sequence 9950 oligonucleotide used to ligate the vectorfragment to create resulting plasmid PS587 18 gatctcccta tagtgagtcgtattaatg 28 19 26 DNA Artificial Sequence 9952-top PCR primer used toisolate the coding sequence of human PKAcat (except the 17 N-terminalamino acids) from human liver or spleen cDNA library 19 gagcgtgaaagaattcttag ccaaag 26 20 29 DNA Artificial Sequence 9922-bottom PCRprimer used to isolate the coding sequence of human PKAcat (except the17 N-terminal amino acids) from human liver or spleen cDNA library 20gtggatccca aaactcagaa aactccttg 29 21 62 DNA Artificial Sequence 9955oligonucleotide which adds remaining N-terminal amino acids to PKAcat 21agcttccgcg atgggcaacg ccgccgccgc caagaagggc agcgagcagg agagcgtgaa 60 ag62 22 62 DNA Artificial Sequence 9956 oligonucleotide which addsremaining N-terminal amino acids to PKAcat 22 aattctttca cgctctcctgctcgctgccc ttcttggcgg cggcggcgtt gcccatcgcg 60 ga 62 23 32 DNAArtificial Sequence 0143-top PCR primer used to isolate the codingsequence of human GRB2 from the PS587 plasmid 23 gttggatccc atggaagccatcgccaaata tg 32 24 29 DNA Artificial Sequence 0142-bottom PCR primerused to isolate the coding sequence of human GRB2 from the PS587 plasmid24 gtttctagat tagacgttcc ggttcacgg 29 25 33 DNA Artificial Sequence 0122PCR primer used to isolate the C-terminal part of the coding sequence ofhuman SOS1 from human fetus or brain cDNA library 25 gttctcgagtcatgagcttt agtcggattg ctg 33 26 33 DNA Artificial Sequence 0123 PCRprimer used to isolate the C-terminal part of the coding sequence ofhuman SOS1 from human fetus or brain cDNA library 26 gttggatcctcattggggag tttctgcatt ttc 33 27 22 DNA Artificial Sequence 9916 PCRprimer used to isolate the Cys1 domain of human PKCgamma from humanbrain cDNA library 27 gtgaattcgg ccatggctgg tc 22 28 28 DNA ArtificialSequence 9935 PCR primer used to isolate the Cys1 domain of humanPKCgamma from human brain cDNA library 28 gtggtacctt cccagcgcct ggacactc28 29 32 DNA Artificial Sequence 4A1-top PCR primer used to amplify thecoding region of HSPDE4A1 29 gtaagcttaa gatgcccttg gtggatttct tc 32

1. A method for detecting if a compound modulates an intracellular protein interaction comprising the steps of: (a) providing a cell that contains two heterologous conjugates, the first heterologous conjugate comprising a first protein of interest conjugated to a detectable group, the second heterologous conjugate comprising a second protein of interest conjugated to an anchor protein that can specifically bind to an internal structure within the cell; (b) detecting the intracellular distribution of the detectable group an intracellular distribution of said detectable group mimicking the intracellular distribution of the anchor-protein being indicative of binding between the two proteins of interest; (c) repeating step (b) with and without the compound; a change in intracellular distribution of the detectable group with and without the compound being indicative of the compound modulating said protein interaction.
 2. A method according to claim 1, wherein the specific binding to the internal structure within the cell is dissolved by addition of an anchor stimulus.
 3. A method according to the previous claim, wherein the specific binding is dissolved by addition of forskolin and wherein the anchor protein is PKA.
 4. A method according to any of the preceding claims, wherein the anchor protein is PKAcata.
 5. A method according to any of the previous claims, wherein the specific binding is dissolved by addition of rolipram and wherein the anchor protein is PDE4A1.
 6. A method according to any of the previous claims, wherein the specific binding is dissolved by addition of ATP and wherein the anchor protein is PLC-delta.
 7. A method according to any of the previous claims, wherein the specific binding is dissolved by addition of Trichostatin A (TSA) and wherein the anchor protein is histonedeacetylase 5 (HDAC5).
 8. A method according to any of the preceding claims, wherein the specific binding to the internal structure within the cell is induced stimulus that by itself has little likelihood of stimulating or inhibiting signalling activity within the cell of interest.
 9. A method according to the previous claims, wherein the specific binding is induced by addition of rolipram and wherein the anchor protein is PDE4A4.
 10. A method according to any of the preceding claims, wherein the anchor protein is Cys1 domain.
 11. A method according to any of the preceding claims, wherein the detectable group is GFP.
 12. A method for measuring a change in mobility of a cellular component caused by an influence, the method comprising: (a) contacting cells with and without the influence, the cells comprising a luminophore coupled to the cellular component; (b) adding extraction buffer to the cells of step (a), the extraction buffer comprising a cellular fixation agent and a cellular permeabilisation agent; and (c) measuring the light emitted from the luminophore from cells of step (b); wherein a difference in light intensity emitted from the cells with and without the influence indicates a difference in the mobility of the cellular component caused by the influence.
 13. A method for measuring a change in mobility of a cellular component according to any of the previous claims, wherein the change in mobility is a change from a soluble cytoplasmic state to an attached cytoplasmic state or vice versa.
 14. A method for measuring a change in mobility of a cellular component according to any of the previous claims, wherein the change in mobility is a change from a soluble cytoplasmic state to a state wherein the component is attached to the cellular membrane or vice versa.
 15. A method for measuring a change in mobility of a cellular component according to any of the previous claims, wherein the change in mobility is a change from a soluble cytoplasmic state to a state wherein the component is attached to, or incorporated in, the nucleus or vice versa.
 16. A method for measuring a change in mobility of a cellular component according to any of the previous claims, wherein the change in mobility is a change from an attached cytoplasmic state to a wherein the component is attached to, or incorporated in, the nucleus or vice versa.
 17. A method according to any of the previous claims, wherein the influence is a chemical substance.
 18. A method according to any of the preceding claims wherein the luminophore is a polypeptide encoded by and expressed from a nucleotide sequence harboured in the cells.
 19. A method according to any of the preceding claims, wherein the luminophore is a hybrid polypeptide comprising a fusion of at least a portion of each of two polypeptides one of which comprises a luminescent polypeptide and the other one of which comprises the cellular component.
 20. A method according to any of the precding claim, wherein the luminescent polypeptide is a Green Fluorescent Protein (GFP).
 21. A method according to any of the preceding claims, wherein the GFP is a GFP having the F64L mutation.
 22. A method according to any of the preceding claims, wherein the GFP is a GFP variant selected from the group consisting of F64L-GFP, F64L-Y66H-GFP, F64L-S65T-GFP, F64L-E222G-GFP and EGFP.
 23. A method for measuring intracellular redistribution according to any of the previous claims, wherein the extraction buffer comprises as the cellular fixation agent formalin.
 24. A method for measuring intracellular redistribution according to any of the previous claims, wherein the extraction buffer comprises as the cellular penneabilisation agent Triton-X.
 25. A method for optimizing the extraction buffer comprising the steps of: (a) contacting or incubating with the reference compound a mechanically intact living cell or mechanically intact living cells comprising a luminophore, the luminophore being capable of being redistributed in a manner which is related to the influence of the substance, and/or of being associated with a component which is capable of being redistributed in a manner which is related to the influence of the substance; (b) contacting or incubating without the reference compound cells similar to the cells in (a); (c) adding extraction buffer, the extraction buffer comprising a cellular fixation agent and a cellular permeabilisation agent to the cells in (a) and (b); (d) measuring the light emitted from the luminophore; (e) repeating steps (a) through (d) with extraction buffers with various concentrations of cellular fixation agent and cellular permeabilisation agent; (f) calculating the signal to noise (s/n) ratio as (fluorescence in stimulated cells minus the fluorescence in non-stimulated cells) divided by the fluorescence in the non-stimulated cells times 100% for each of the extraction buffers tested in step (e); the optimized extraction buffer being the buffer associated with the highest signal to noise ratio.
 26. An extraction buffer comprising a cellular fixation agent and a cellular permeabilisation agent.
 27. An extraction buffer according to any of the previous claims, wherein the extraction buffer comprises as the cellular fixation agent formalin.
 28. An extraction buffer according to any of the previous claims, wherein the extraction buffer comprises as the cellular perneabilisation agent Triton-X. 